PmtA Regulates Pyocyanin Expression and Biofilm Formation in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa expresses a small molecular weight, cysteine-rich protein (PmtA), identified as a metallothionein (MT) protein family member. The MT family proteins have been well-characterized in eukaryotes as essential for zinc and copper homeostasis, protection against oxidative stress, and the ability to modify a variety of immune activities. Bacterial MTs share sequence homology, antioxidant chemistry, and heavy metal-binding capacity with eukaryotic MTs, however, the impact of bacterial MTs on virulence and infection have not been well-studied. In the present study, we investigated the role of PmtA in P. aeruginosa PAO1 using a PmtA-deficient strain (ΔpmtA). Here we demonstrated the virulence factor, pyocyanin, relies on the expression of PmtA. We showed that PmtA may be protective against oxidative stress, as an alternative antioxidant, glutathione, can rescue pyocyanin expression. Furthermore, the expression of phzM, which encodes a pyocyanin precursor enzyme, was decreased in the ΔpmtA mutant during early stationary phase. Upregulated pmtA expression was previously detected in confluent biofilms, which are essential for chronic infection, and we observed that the ΔpmtA mutant was disrupted for biofilm formation. As biofilms also modulate antibiotic susceptibility, we examined the ΔpmtA mutant susceptibility to antibiotics and found that the ΔpmtA mutant is more susceptible to cefepime and ciprofloxacin than the wild-type strain. Finally, we observed that the deletion of pmtA results in decreased virulence in a waxworm model. Taken together, our results support the conclusion that PmtA is necessary for the full virulence of P. aeruginosa and may represent a potential target for therapeutic intervention.


INTRODUCTION
Pseudomonas aeruginosa is a non-fermenting Gram-negative, opportunistic pathogen that ranks amongst the top five hospital-associated infections. Most commonly, these bacteria are found in patients that are immunocompromised, have undergone invasive surgery and/or have underlying conditions such as diabetes (Moradali et al., 2017;Wong et al., 2021). P. aeruginosa infections frequently arise from associations with medical devices such as ventilators, central lines, urinary catheters and/or surgical/transplantation (Moradali et al., 2017) and have been implicated as the causative agent of a wide variety of infections (e.g., pneumonia, sepsis, keratitis, skin, bone, joint, endocarditis, and meningitis) (Gellatly and Hancock, 2013;Magalhães et al., 2016;Wong et al., 2021). P. aeruginosa infections associated with chronic lung disease such as cystic fibrosis disease, ventilator-associated pneumonia (VAP), and individuals with chronic obstructive pulmonary disease (COPD) are persistent and are often linked with an increase in morbidity and mortality (Moradali et al., 2017;Malhotra et al., 2019). More recently, P. aeruginosa has been found as a common coinfection in patients hospitalized with COVID-19 (Qu et al., 2021). A major challenge in effectively treating these infections is multidrug-resistant (MDR) strains of P. aeruginosa. MDR strains have been associated with increased visits to clinical settings, very poor clinical outcomes, and are considered critical on the priority list by the World Health Organization (Tacconelli et al., 2013;Shrivastava and Ramasamy, 2018;Malhotra et al., 2019). Intrinsic antimicrobial resistance genes make P. aeruginosa highly resistant to antibiotics. These genes include efflux pump systems, beta-lactamase production, porin alterations, and target site modifications (Chuanchuen et al., 2002;Aedekerk et al., 2005;Reygaert, 2018). Although conventional antibiotics such as cefepime and ciprofloxacin are currently used to treat P. aeruginosa infections, resistance rates continue to rise in the four major groups of anti-pseudomonal agents: carbapenem, aminoglycosides, cephalosporins, and fluoroquinolones (Marvig et al., 2014;Pachori et al., 2019;Gajdács et al., 2021). Therefore, without reasonable availability of new therapeutic agents, drug resistance may worsen. Recently, targeting virulence factors has emerged as a new line of action in combating MDR P. aeruginosa strains and has revealed 12 classes of smallmolecule inhibitors and two antibodies that attack key virulence regulators (Shao et al., 2020).
To establish an infection, P. aeruginosa uses an array of virulence factors to counteract host defenses, cause direct damage to the host tissues and increase microbial competitiveness (Gellatly and Hancock, 2013). Some of these virulence factors involve phenazines, biofilms, exotoxins, endotoxins, proteases, siderophores, flagella, and secretion systems (Behzadi et al., 2021). Phenazines are secondary metabolites produced by Pseudomonas species that have been shown to increase microbial virulence (Das et al., 2016;Gonçalves and Vasconcelos, 2021). P. aeruginosa synthesizes a variety of these compounds, i.e., pyocyanin, phenazine-1carboxylic acid (PCA), 1-hydroxyphenazine (1-OH-PHZ) and phenazine1-carboxamine (PCN). Construction of phenazines involves two homologous core loci biosynthetic pathways (phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2) along with three genes (phzM, phzS and phzH) that encode enzymes responsible for converting PCA to pyocyanin, PCN and 1-OH-PHZ. Pyocyanin is a well-characterized virulence factor produced by 95% of P. aeruginosa isolates (Gonçalves and Vasconcelos, 2021). Importantly, this oxidant-sensitive metabolite allows P. aeruginosa to manipulate the redox micro-environment during infection. Pyocyanin induces reactive oxygen species (ROS) production by host cells and blocks the host expression of catalase, an enzyme that neutralizes ROS through NADPH oxidation (Lau et al., 2004a,b). Sustained exposure to ROS results in host cell damage and a decrease in host defenses, which allows bacteria to establish a chronic infection (Gellatly and Hancock, 2013). In a murine infection model, pyocyanin-deficient mutants are more readily cleared from the lungs and less able to cause pneumonia than their wild-type counterparts (Lau et al., 2004b). Taken together, these results suggest that pyocyanin expression is essential for disease.
Another important virulence factor for P. aeruginosa is the ability to form biofilms. Biofilms are complex communities of microbes encapsulated in an extracellular polymeric substance (EPS) matrix that is comprised of proteins, lipids and extracellular DNA. Extracellular DNA (eDNA) has been identified as a major component of P. aeruginosa biofilm that is released by the lysing of a subpopulation of P. aeruginosa cells. In P. aeruginosa, eDNA release is mediated by quorum sensing (QS) signaling molecules N-acyl-L-homoserine lactone and Pseudomonas quinolone. Mediation of eDNA can also be affected by flagella and type IV pili mechanisms. In addition to initiating biofilm formation, eDNA can also bind to antibiotics, affording biofilm-associated cells even greater protection from these molecules. Pyocyanin is also regulated by QS and is essential for the release of eDNA, aiding in the formation of biofilms (Das et al., 2013).
Pseudomonas aeruginosa expresses a metallothionein (MT) known as PmtA (P. aeruginosa MT A) (Blindauer, 2008). Although little is known regarding the function of bacterial MTs, they do share some sequence homology, heavy metal binding characteristics, and cysteine motifs with their eukaryotic counterparts ( Figure 1A and Table 1). Eukaryotic MTs are well studied in mammals for their immunomodulatory role. MTs can influence leukocyte trafficking and decrease proinflammatory mediators (Youn et al., 1995;Yin et al., 2005). Additionally, MT expression intensifies inflammatory bowel disease, alters the antibody response to antigen challenge, and anti-MT antibodies can ameliorate the severity of chronic inflammation (Lynes et al., 1993;Laukens et al., 2009;Devisscher et al., 2014).
A classic feature of the MT protein family is an abundance of cysteine residues and the presence of free thiol groups. These allow MTs to bind divalent heavy metals and contribute to their immunomodulatory functions (Lynes et al., 1993). MTs protects the host from ROS-induced damage during oxidative stress through oxidation of thiol-groups, which may allow macrophages to produce more ROS in the presence of MTs (Borghesi and Lynes, 1996). PmtA may have similar immunomodulatory properties and potentially influence Pseudomonas virulence due to its similarities in structure to mammalian MT. PmtA has also been shown to be upregulated in confluent biofilms compared to planktonic cells (Hentzer et al., 2005;Waite et al., 2006).
To date, the function of PmtA in P. aeruginosa infections in relation to virulence is unknown. In this study, we investigated the role of PmtA in P. aeruginosa strain PAO1, using a clean deletion mutant ( pmtA). We also investigated the role FIGURE 1 | Comparison of PAO1 PmtA and Homo sapiens Metallothionein I and generation valuation of the pmtA mutant. (A) MT from P. aeruginosa PAO1 contains 79 amino acids, 10 of which are cysteine residues. A comparison of human MT-1 (Accession: AAH29475) and PAO1 PmtA (Accession: NP_250830) using the BLAST online software reveals there is little homology between the sequences (e = 6), but the alignment reveals many of the fully conserved regions (*, below sequence) are cysteine residues. A (:) indicates conservation of amino acids with strongly similar properties, while (.) indicates conservation of amino acids with somewhat similar properties. Cysteine residues that are conserved between MT-1 and PmtA are indicated by (*) above the sequence. (B) Confirmation of clean deletion mutant was revealed by colony PCR. Lane 1, shows a 100 bp ladder; lane 2, shows wild-type PAO1 (485 bp), and lane 3, shows the pmtA mutant (263 bp). (C) Overnight cultures of P. aeruginosa strains were grown in LB media for 24 h and total RNA was extracted from the cell pellets. Relative expression values for pmtA were calculated using the cycle threshold value compared to a standard curve for pmtA. pmtA expression was decreased in the pmtA mutant. This expression was restored in the complemented strain, pmtA:pmtA. One-way ANOVA analysis was performed (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments. PmtA plays in biofilm formation, pyocyanin production and antibiotic sensitivity.

MATERIALS AND METHODS
Bacterial Strains, Plasmids, Growth Media, and Antibiotics The bacterial strains, plasmids, and primers used in the present study are listed in Supplementary Tables S1, S2.

Deletion and Complementation of PmtA
An unmarked deletion mutant ( pmtA) was derived from PAO1 using a two-step allelic exchange as previously described (Hoang et al., 1998;Hmelo et al., 2015) and depicted in Supplementary Figure S1. Briefly, using the primer pairs 1_PA2140_del_F/R and 2_PA2140_del_F/R, approximately 500-bp fragments upstream and downstream of the target genes were amplified with 15-20 bp overlapping end complimentary to BamHI-HF (New England BioLabs (NEB), Ipswich MA, R3136S) and EcoRI-HF (NEB, R3101S)-digested pEX18Gm using Q5 High-Fidelity DNA Polymerase (NEB, M0491S). The mutant gene fragments were assembled using Gibson assembly (NEB, E5510S) with double-digested pEX18Gm and transformed into One Shot PIR1 (Invitrogen, Waltham, MA, United States, C101010) chemically competent Escherichia coli cells. Transformants were selected on LB plates supplemented with 15 µg/mL gentamicin and screened using colony PCR with the primer pair 2_PA2140_test_F/R. Clones bearing allelic exchange vectors, in which the mutant allele was inserted, were then assessed for correctness using Sanger sequencing. Plasmids were purified, transformed into donor E. coli strain SM10 λ-pir and conjugated into P. aeruginosa PAO1 recipient cells. Transformants were selected on Vogel-Bonner minimal media containing 10 mM citrate as a carbon source to select against E. coli. To select against merodiploids, LB agar containing 15% (wt/vol) sucrose and no NaCl 2 was used for counterselection. Pseudomonas isolation agar containing 60 µg/mL gentamicin was used to select for isolates having undergone spontaneous excision of the plasmid. Isolates that no longer could grow in the presence of gentamicin were screened for deletion of the wild-type allele using the primer pair PA2140_Tn7_F/R by colony PCR ( Figure 1B) and assessed for correctness by Sanger sequencing. A complemented deletion mutant strain ( pmtA:pmtA) was generated with pUC18R6K-mini-Tn7T-Gm (Addgene, Watertown, MA, United States, 65022) as described by Choi and Schweizer (2006). The full-length of the target gene (including a potential promoter) was amplified from P. aeruginosa strain PAO1 using the primer pairs PPA2140_Tn7_F/PA2140_Tn7_R. Amplification fragments were assembled using Gibson assembly with PstI-HF (NEB, R3140S) and KpnI-HF (NEB, R3142S)digested pUC18R6K-mini-Tn7T-Gm and transformed into One Shot PIR1 chemically competent E. coli cells for directed recombination of the fragment into the plasmid. The pmtA mutant was transformed with the suicide plasmid and selected for on LB medium containing 60 µg/mL of gentamicin. Isolates that grew in the presence of gentamicin were screened for the presence of the complementation construct using the primer pair pTn7GM_F/R by colony PCR (Supplementary Figure S4) and assessed for correctness by Sanger sequencing.

Bacterial Growth
Overnight cultures were diluted 1:100 in fresh LB or M9 salt media without antibiotics, diluted and aliquoted into a 96-well plate (Greiner Bio-One, Monroe, NC, United States, 655101) in triplicate and then monitored for growth by measuring the optical density at 600 nm (OD 600 ) in a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA, United States). As P. aeruginosa aggregates after the first 4 h of cultivation, the strains were diluted 1:10 in LB media and vortexed before the OD 600 was determined. For P. aeruginosa growth curves in LB supplemented with oxidants and metals, overnight cultures were diluted 1:100 in fresh LB medium and grown for 2 h to exponential phase. Then, 600 µL of each culture was added in triplicate to the wells of a 24-well sterile plate (Fisher Scientific, Waltham, MA, United States, Cat#353047) together with 10 mM hydrogen peroxide, 100 µM CdCl 2 , 200 µM ZnCl 2 , or medium. The strains were cultured for 10-12 h with intermittent shaking at 37 • C, after which growth was calculated by dividing the average OD 600 value of triplicate cultures grown with oxidant by that obtained for cultures grown without oxidant. For M9 salt medium, overnight cultures were subcultured to an OD 600 of 0.08, and 600 µL was added to the wells of a 24-well sterile plate (Falcon, Cat#353047) in triplicate and mixed with 400 µL of M9 salt medium alone or supplemented with oxidants or metals (200 µg/mL transferrin, 150 µM ethylenediaminediacetic acid (EDDA), 50 µM FeCl 2 , 10 mM hydrogen peroxide, 200 µM CdCl 2 , 100 µM ZnCl 2 ). The strains were cultured for 18 h with intermittent shaking at 37 • C. To measure pyocyanin production, single colonies of the P. aeruginosa strains were grown overnight in 5 mL of LB medium with the appropriate antibiotics at 37 • C with vigorous shaking. These cultures were then diluted at 1:100 in fresh LB media without antibiotics at a dilution of 1:100 and grown at 37 • C with vigorous shaking. Pyocyanin concentrations were determined as previously described (Frank and Demoss, 1959) with the exception that 1 mL of each culture was centrifuged for 1 min at 20,500 × g. The supernatants were transferred to new microcentrifuge tubes, vortexed vigorously and then transferred to a 96-well plate in triplicate to measure the absorbance at 691 nm, with measurements taken at selected time points over 30 h of cultivation. The OD 600 values of the cultures were also monitored, and data was plotted as OD 600 vs. Abs 691 .

Pyocyanin Extraction
Pseudomonas aeruginosa strains were grown for 30 h in triplicate, and 1 mL of cell-free supernatants were obtained. Pyocyanin was extracted as previously described with minor modifications (Frank and Demoss, 1959;Essar et al., 1990;Zaborin et al., 2012). In brief, 500 µL of chloroform was added to 1 mL of supernatant from each strain. The mixture was then vigorously vortexed for 2 min as the chloroform turned blue green, after which the tubes were centrifuged for 10 min at 3000 × g and 4 • C. Hydrochloric acid (150 µL, 0.2 M) was added to the partially extracted blue pyocyanin and vortexed vigorously for 2 min. The top blue layer was acidified to a pink color and samples were centrifuged for 2 min at 3000 × g and 4 • C. The absorbance of completely extracted pyocyanin at the pink stage was measured at 520 nm. Measurements were normalized to initial cell density OD 600 .

RT-qPCR
Pseudomonas aeruginosa strains were grown overnight in LB medium to stationary phase and a subculture was used to collect total RNA extracted from the cell pellets using QIAGEN RNeasy kit (74104) according to the manufacturer's recommendations at 4, 10, and 24 h. RNA was converted to cDNA using the TaqMan Reverse Transcription kit (Invitrogen, N8080234). Gene-specific primers to pmtA and the housekeeping ribosomal gene (RNA polymerase, subunit alpha) were designed and are listed in Supplementary Table S2. qPCR was performed on an Applied Biosystems 7300 instrument using iTaq Universal SYBR green (Bio-Rad, Hercules, CA, United States, 1725121). All qPCR experiments were performed with at least three biological replicates, each tested in triplicate for each time point. The absolute gene expression for pmtA was calculated using the cycle threshold value compared to a standard curve of pmtA using pGEX-6p-pmtA. The qPCR assay efficiency was verified using a five-fold dilution standard curve in triplicate. The R 2 of the standard curve was 0.99 and efficiency was 100%. qPCR was performed on an Applied Biosystems 7300 instrument using iTaq Universal SYBR green (Bio-Rad, 1725121). All qPCR experiments were performed with at least three biological replicates, each tested in quadruplicate for each timepoint. The relative gene expression for phzH and phzM was calculated using the cycle threshold value compared to the ribosomal RNA gene as the internal reference standard. Fold changes were expressed as 2 − Ct values. No template and wild-type PAO1 were used negative and positive control, respectively.

Biofilm Formation Assay
The wild-type, pmtA, and pmtA:pmtA PAO1 strains were inoculated into 5 mL of LB and vigorously shaken at an angle in 14-mL Falcon tubes overnight. The medium was removed the next day, and the splash zone was stained for 15 min with 0.1% crystal violet (Fisher Science, Waltham, MA, United States, S25275A). Subsequently, the tubes were gently rinsed with water (2×) and inspected for the presence of a biofilm. Biofilm formation was also quantified using a previously described microtiter plate assay with minor adaptations (O'Toole, 2010). In brief, overnight cultures were diluted at 1:10 in fresh LB medium and grown for 24 h at 37 • C without shaking. Growth yields were determined by measuring the absorbance at 595 nm. The medium was removed, and the biofilms were fixed to the sides of the microtiter wells with 90% methanol before being stained with 0.1% crystal violet (Fisher Science, Waltham, MA, United States, S25275A). The crystal violet was solubilized in ethanol:acetone (4:1, v/v) and measured at 595 nm using an ethanol solution as blank. P. aeruginosa PAO1 was used as a positive control, with three biological replicates and eight technical replicates performed for each strain. The relative biofilm formation (RBF) for each well was determined by dividing the crystal violet absorbance reading by the growth yield absorbance reading.

Acyl Homoserine Lactone-Dependent Quorum Sensing Assay
The reporter strain E. coli MG4 pKDT17 (Addgene, Watertown, MA, United States, 27503) was used in crossfeeding assays with the wild-type, pmtA or pmtA:pmtA PAO1 strains and E. coli MC1061 (negative control) to assess acyl homoserine lactone (AHL) production as previously described (Pearson et al., 1995;Van Laar et al., 2018). In brief, wild-type, pmtA, and pmtA:pmtA PAO1 strains and E. coli MG4 were grown overnight. Subsequently, each PAO1 strain was streaked onto a MacConkey agar plate 0.75 cm from a streak of E. coli MG4 and incubated overnight at 37 • C. The next day, the plates were examined for color change, where pink or purple indicated the presence of AHLs and white indicated their absence.

Minimum Inhibitory Concentration Determination Using E-Test
The susceptibility of P. aeruginosa strains to ciprofloxacin, cefepime and tetracycline, was assessed using Liofilchem R MTS TM E-tests (Fisher Scientific,Waltham,MA,United States, following the manufacturer's protocol. In short, fresh P. aeruginosa colonies were resuspended in 1 mL of LB, and a cotton swab was then used to spread the inoculated LB over LB agar plates. The plates were allowed to dry, and then the E-test strip was then placed onto the inculcated agar, with each strain assayed in triplicate. The plates were incubated overnight at 37 • C, and the minimum inhibitory concentration (MIC) was read the next day.

In vivo Galleria mellonella Larvae Model of Infection
Healthy Galleria mellonella larvae (waxworms) were purchased from Vanderhorst Wholesale, Inc. (St Marys, OH, United States) and briefly stored at 4 • C until use. Injections were performed as described in Seed and Dennis (2008) with a few adaptations. Cultures of P. aeruginosa strains were grown overnight, and the ability of 10 3 CFU/mL of each P. aeruginosa to cause melanization was assessed. Worms were randomly separated into groups and injected into the hindmost left proleg with 5 µL of sterile 10 mM MgSO 4 or 5 µL of 10 3 CFU/mL of the wild-type pmtA or pmtA:pmtA PAO1 strains using a 10-µL Hamilton syringe (Hamilton Company, Reno, NV, United States) with a beveled needle. The inoculum (10 3 CFU/mL) was verified by plating, and the injected worms were incubated at 37 • C 48 h, with survival assessed at 20, 24, 28, and 48 h post injection (Tsai et al., 2016).

The pmtA Clean Deletion Mutant Has no Detectable Metallothionein Production
Despite being highly conserved, little is known regarding the physiological function and expression of pmtA during the life cycle of P. aeruginosa. We constructed a clean deletion mutation of pmtA in wild-type P. aeruginosa PAO1 using a two-step allelic exchange method as well as a complemented strain ( pmtA:pmtA) by cloning pmtA in a Tn7 and conjugating it into pmtA. We verified the genetic change in the pmtA strain using colony PCR and Sanger sequencing ( Figure 1B,  Supplementary Figure S2 and data not shown). The colony PCR shows a band shift when comparing the products from the PAO1 (485 bp) and pmtA (263 bp) strains, indicating success in generation of a clean mutation. RT-qPCR was also used to assess pmtA transcription levels in the wild-type, pmtA and pmtA:pmtA PAO1 strains. The wild-type and pmtA:pmtA strains had approximately equal amounts of transcript, while little to no pmtA transcript was present in pmtA mutant cultures at 4, 10, and 24 h ( Figure 1C). These results also suggest that pmtA is expressed throughout stationary phases in PAO1.
The pmtA, and pmtA:pmtA PAO1 Strains Have Similar Growth Kinetics The growth kinetics of the wild-type, pmtA, and pmtA:pmtA PAO1 strains were examined in both LB and M9 salt media. While no significant differences in the growth rates were detected between the strains (Supplementary Figures S3A-C), the wildtype and pmtA:pmtA strains turned the media green after 10 h of growth, while the pmtA culture media remained yellow (Figure 2A). This color difference persisted to 30 h of growth and is likely due to a difference in the production of pyocyanin, which is known to be secreted by PAO1 and imparts a distinct bluish-green color to media (Hassan and Fridovich, 1980).

The pmtA Mutant Produces Less Pyocyanin Than Wild-Type PAO1
We investigated the ability of the pmtA mutant to produce pyocyanin by determining the absorbance values of cell-free culture supernatants at 691 nm in a time course assay (Rada and Leto, 2013). Pyocyanin levels were decreased in the culture supernatants of the pmtA strain compared to the wild-type and pmtA:pmtA strains starting at 10 h and remained lower than those of the wild-type strain throughout the assay (Figure 2B). The highest levels of pyocyanin expression by PAO1 are observed during stationary phase growth (Rada and Leto, 2013). We compared cell growth to pyocyanin levels (691/600 nm ratio) for the various PAO1 strains, and the normalized pyocyanin levels were consistently higher in the wild-type and pmtA:pmtA than in the pmtA mutant ( Figure 2C). QS molecules, phenazineproducing genes (phzA-G), and the gene phzM are involved in pyocyanin production at early stationary phase. Wild-type PAO1 starts to produce detectable pyocyanin at 10 h (Das et al., 2016; Figures 2B,C). These results indicate that pmtA is necessary for optimum pyocyanin levels in P. aeruginosa and that the restoration of pmtA expression is sufficient for re-establishing pyocyanin to normal levels.
Pyocyanin appears as different colors in media depending on the pH and oxidation status of the culture (Rada and Leto, 2013). Highly oxygenated cultures are green due to the accumulation of oxidized pyocyanin, whereas those that are not well oxygenated appear yellow due to reduced pyocyanin (Rada and Leto, 2013). To ensure that the 691/600 nm ratio of wild-type PAO1 was not higher than that of the pmtA mutant due to a difference in the pH or oxidation status, pyocyanin was extracted with chloroform/HCl after 24 h of growth, and the absorption readings of the supernatants were analyzed as previously described (Rada and Leto, 2013). Wildtype PAO1 extracts were significantly higher than the pmtA mutant extracts, while extracts were restored to wild-type levels in the complemented mutant pmtA:pmtA ( Figure 2D). These results indicate that wild-type PAO1 produces more pyocyanin than the pmtA mutant and the difference in the 691 nm readings is not due to a difference in the oxidation state of pyocyanin or the pH of the culture.
Exogenous Addition of the Antioxidant Glutathione Restores Pyocyanin Production to Wild-Type Levels in the pmtA Mutant Because the pmtA mutant produces less pyocyanin, we hypothesize that this strain also has a reduction in antioxidant capacity. To test this hypothesis, the alternative antioxidant glutathione (GSH) was added to the growth media, and changes in pyocyanin production were monitored. The absorbance values at 691 and 600 nm were measured at 0, 2, 4, 6, 8, 10, 12, 24, and 30 h of growth and the 691/600 nm ratios were calculated ( Figure 3A). The addition of biological levels of GSH restored pyocyanin to wild-type levels, indicating that PmtA alters the redox environment and influences pyocyanin production ( Figure 3B; Meister and Anderson, 1983).

PmtA Plays an Essential Role in Phenazine Production
The phzA1 and phzA2 gene clusters encode enzymes required for phenazine synthesis to produce phenazine-1-carboxylic acid. PhzH converts phenazine-1-carboxylic acid to phenazine-1carboxamide, while PhzM converts phenazine-1-carboxylic acid to 5-methylphenazine-1-carboxylic acid betaine, which is later converted to pyocyanin by PhzS (Mavrodi et al., 2001). To elucidate the mechanism by which PmtA affects pyocyanin biosynthesis, we assessed the expression of phzH and phzM using real-time PCR (Figure 4). The expression of phzM but not phzH was decreased in the pmtA mutant (Figure 4A), and phzM expression was restored in the complemented mutant pmtA:pmtA at 4 h. However, no differences were observed between the wild-type, pmtA and pmtA:pmtA strains at the later time points (10 and 24 h). These results suggest that PmtA biosynthesis is required for the early expression of phzM but not phzH via unknown mechanisms and that the expression levels of this gene contribute to the overall production of the pyocyanin precursor 5-methylphenazine-1-carboxylic acid betaine in P. aeruginosa.

PmtA Is Essential for Biofilm Formation
As upregulated PmtA expression has been observed in confluent P. aeruginosa biofilms (Hentzer et al., 2005;Waite et al., 2006), we investigated the role of PmtA in biofilm formation. First, we assessed biofilm formation by the wild-type, pmtA, and pmtA:pmtA PAO1 strains by examining the splash zones of overnight cultures stained with crystal violet. As shown in Figure 5A, distinct crystal violet-stained biofilms were formed by the wild-type and pmtA:pmtA strains but not the pmtA mutant. Additionally, a crystal violet microtiter plate biofilm assay was used to quantitate the biofilm formation defect FIGURE 2 | P. aeruginosa PAO1 strain pmtA produces less pyocyanin than wild-type PAO1. (A) P. aeruginosa strains were each grown overnight in 5 mL of LB medium at 37 • C for 24 h with vigorous shaking. The cultures were observed for color change and the pmtA mutant had a yellow appearance compared to wild-type PAO1. (B) Pyocyanin levels were measured at 0, 2, 4, 6, 8, 10, 12, 24, and 30 h using 1 mL of the culture and centrifuged for 1 min at 20,500 × g. The supernatant was then placed in a 96 well plate in triplicate and the absorbance at 691 nm was measured. (C) The data was plotted as OD 600 vs. OD 691 . (D) P. aeruginosa strains were grown for 24 h, and cell-free supernatants were obtained. Pyocyanin was extracted as previously described, with minor modifications (Essar et al., 1990;Zaborin et al., 2012). Chloroform was added to the supernatant from each strain, forming a blue bottom layer. Subsequently, hydrochloric acid (1.5 mL, 0.2 M) was added to the layer altering the pH to 2 and pink in color. The absorbance of completely extracted pyocyanin was measured at OD 520 and the pyocyanin/cell density was calculated by dividing the pyocyanin at OD 520 by growth measured at OD 600 . One-way ANOVA analysis was performed (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
presented by the pmtA strain. Interestingly, the pmtA mutant showed a 28% decrease in biofilm formation compared to the wild-type and pmtA:pmtA PAO1 strains ( Figure 5B). These results indicate that the lack of PmtA contributed to decreased biofilm production by the pmtA mutant and that the restoration of PmtA production resulted in the reestablishment of biofilm formation. Incidentally, the addition of the antioxidant GSH restored biofilm formation in the pmtA mutant, confirming that redox plays a role in P. aeruginosa biofilm formation (Figure 5C).

PmtA Is Not Essential for Managing Oxidative or Metal Stress
Low-molecular-weight thiols are essential for detoxifying oxidative stressors and ameliorating heavy metal stress (Van Laar et al., 2018;Ulrich and Jakob, 2019). For example, the lowmolecular-weight thiol OspR can act as an oxidative stress sensor in P. aeruginosa. MTs can actively scavenge reactive oxygen and nitrogen species due to their high abundance of cysteine residues (Ruttkay-Nedecky et al., 2013). In a previous study, pmtA expression was shown to be upregulated upon 10 mM H 2 O 2 stimulation for 15 min compared to untreated controls (Goldová et al., 2011). Therefore, we assessed whether the lack of PmtA contributes to increased sensitivity to various oxidants and heavy metals. First, we characterized the growth of the pmtA mutant compared to the wild-type and pmtA:pmtA PAO1 strains in the presence of 10 mM H 2 O 2 . All three strains grew to similar levels in both LB and M9 salt media supplemented with 10 mM H 2 O 2 (Supplementary Figure S4). These results indicate that PmtA alone does not detoxify peroxides and that other factors may contribute to this process. pmtA:pmtA and pmtA supplemented with 10 mM glutathione (GSH) PAO1 strains were cultured for 30 h at 37 • C with vigorous shaking. Over the 30-h, the growth of the bacteria (OD 600 ) was compared to the production of pyocyanin in media (OD 691 ). The wild-type and pmtA:pmtA PAO1 strains produced more pyocyanin when compared to the pmtA mutant. Pyocyanin production of the pmtA mutant was restored to near wild-type levels starting at 8 h of growth. (B) The wild-type, pmtA, pmtA:pmtA, pmtA and pmtA supplemented with 10 mM glutathione (GSH) PAO1 strains were cultured in LB medium for 30 h at 37 • C with vigorous shaking. At 30 h, pyocyanin was measured in the media (OD 691 ). The wild-type and pmtA:pmtA strains produced more pyocyanin when compared to the pmtA mutant. The pyocyanin production of the pmtA mutant was significantly increased with the addition of GSH. An unpaired t-test analysis was performed (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
Zinc functions as a cofactor for many essential enzymes in all organisms. However, as excess zinc can be toxic, bacteria maintain intracellular zinc concentrations at low levels (Blindauer, 2015). Cadmium is toxic to most organisms and generates oxidative stress (Maret and Moulis, 2013). MTs play a role in zinc and copper homeostasis as well as cadmium detoxification (Blindauer, 2011). Cyanobacterial MTs are encoded within defined operons that include a zinc-dependent repressor (SmtB) and an operator promoter region in the intergenic region (Morby et al., 1993;Blindauer, 2008Blindauer, , 2011. However, Pseudomonas MT-harboring operons appear to lack such features and encode proteins of unknown functions ( Figure 1A and Table 1). In the present study, we investigated the role of PmtA in heavy metal resistance by culturing the wild-type, pmtA, and pmtA:pmtA PAO1 strains in LB and in M9 salt media supplemented with zinc or cadmium and assessing their viability over time (Supplementary Figure S5). All strains grew to similar levels in both media types, with no differences observed between the strains exposed to inhibitory concentrations of ZnCl 2 FIGURE 4 | PmtA is required for the early expression of the phenazine pigment precursor 5-methylphenazine-1-carboxylic acid betaine. Overnight cultures of P. aeruginosa strains were grown in LB medium for 4, 10, and 24 h, and total RNA was extracted from the cell pellets. RNA was converted to cDNA using a TaqMan Reverse Transcription kit. qPCR was performed on an ABI7300 instrument using SYBR green fluorescent dye and ROX as a detector. The relative gene expression values for phzM (A-C) and phzH (D-F) were calculated using the cycle threshold value compared to the RNA polymerase, subunit alpha. (A) The expression of phzM at 4 h decreased in the pmtA mutant and was restored in the complemented mutant pmtA:pmtA. CT (cycle threshold) values were used to calculate RQ (relative quantitation) values for phzH and phzM at each time point and normalized to the housekeeping gene RNA polymerase. An unpaired t-test analysis (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
(200 µM) (Supplementary Figures S5A,B) or CdCl 2 (100 µM) (Supplementary Figures S5C,D). In addition, no differences were observed between the strains when cultured for 6 h before exposure (data not shown). The similar growth pattern of the strains under zinc or cadmium excess suggests that PmtA is not crucial for metal resistance.
As iron uptake is also associated with pyocyanin production, we examined the ability of the wild-type, pmtA, and pmtA:pmtA strains to grow iron chelator EDDA. All strains had a reduced growth rate when compared to growth in M9 salt media indicating the iron limiting conditions. FeCl 2 and transferrin restored the growth of all the stains showing that iron utilization is not affected by the loss of PmtA (Supplementary Figure S6).

PmtA Does Not Inhibit Acyl Homoserine Lactone (AHL)-Mediated Quorum Sensing
Quorum sensing has been shown to mediate many processes in P. aeruginosa, including pyocyanin production and biofilm formation. Therefore, we assessed whether AHL-dependent QS is altered in the pmtA mutant using the reporter strain E. coli MG4 (pKDT17). In this strain, LasR transcribes a lasB-lacZ gene fusion in the presence of AHLs, and the resulting β-galactosidase fusion protein generates a color change in colonies from white to pink/purple when streaked on MacConkey agar. The wildtype, pmtA, and pmtA:pmtA PAO1 strains all induced a color change when streaked on MacConkey agar, indicating that AHL was produced and that PmtA does not mediate AHL-dependent QS (Figures 6A-C). As a negative control, strain E. coli MC1061 was also assessed, no color change was observed ( Figure 6D).

PmtA Promotes Antibiotic Resistance in PAO1
Weakened resistance to antibiotics is a classic biofilm defect characteristic. Therefore, we examined the sensitivity of the pmtA mutant to tetracycline, to which P. aeruginosa is intrinsically resistant, and two of the conventional antibiotics used to treat CF patients infected with P. aeruginosa, cefepime and ciprofloxacin. Using test strips, we quantitatively assessed the MIC of the antibiotics on the wild-type, pmtA, and FIGURE 5 | PmtA is required for biofilm formation and the addition of antioxidant glutathione restores biofilm formation in P. aeruginosa pmtA mutant. (A) Cultures of P. aeruginosa strains were grown overnight in LB medium and stained with crystal violet. Less crystal violet was observed in the pmtA mutant when compared to wild-type and pmtA:pmtA PAO1 strains. (B) Cultures of P. aeruginosa strains were grown overnight in LB media, diluted 1:20 in fresh LB medium and grown to 0.5 OD 600 . After 24 h of growth, growth yield was measured at OD 600 . Biofilms were stained as previously described with minor modifications (O'Toole, 2010) and crystal violet stain was measured at OD 595 . The OD reading after the addition of crystal violet was divided by the growth yield OD to calculate the relative biofilm formation. (C) GSH at 10 mM was added to the LB media in the 96 well plate assay to determine if an antioxidant would rescue the biofilm formation defect in the pmtA mutant. GSH restored the biofilm formation of the pmtA mutant in the microtiter plate assay. An unpaired t-test analysis was performed (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
pmtA:pmtA strains (Figure 7). Compared to the wild-type and pmtA:pmtA PAO1 strains, the pmtA mutant exhibited significantly lower MICs for cefepime and ciprofloxacin, indicating that PmtA promotes resistance to these antibiotics. However, no differences were observed among strains exposed to tetracycline, suggesting that PmtA does not affect P. aeruginosa growth in the presence of this antibiotic, to which it is naturally resistant.

PmtA Expression Influences Pseudomonas aeruginosa Virulence in vivo
The correlation between PmtA expression and the crucial virulence factors pyocyanin and biofilm formation indicates that PmtA may contribute to the overall virulence of P. aeruginosa. To evaluate the virulence of the pmtA mutant, we used G. mellonella larvae, a model system used to test the virulence of infectious bacteria and the innate immune response to cope with infection (Hoffmann, 1995;Mulcahy et al., 2011;Haller et al., 2014). Larvae were injected with 5 µL of diluted cultures containing 10 3 cells of the wild-type. pmtA, or pmtA:pmtA strains. Survival of the larvae was evaluated 20, 24, 28 and 48 h after injection (Figure 8). At 20 h, 100% of the larvae injected with the pmtA mutant survived compared to 10% of those injected with the wild-type strain, a 90% difference in survival rates. Compared to the pmtA mutant, the larvae injected with the complemented strain showed a 42% decrease in survival at 20 h. At 24 h, the percent survival of larvae injected with the pmtA mutant decreased to 52.5%, while those injected FIGURE 6 | PmtA does not affect acyl homoserine lactones levels. Wild-type (A), pmtA (B), and pmtA:pmtA (C). PAO1 (D) E. coli MC1061 strains were streaked out on MacConkey agar plates 0.75 cm apart from E. coli strain MG4. In the presence of AHL, lasR initiates the transcription of LasB-LacZ fusion protein resulting in beta-galactosidase in MG4, this causes colonies to change color from white to pink. All three strains caused the color change to pink suggesting that QS mediated by AHL is not inhibited by the absence of PmtA. pmtA, and pmtA:pmtA PAO1 strains were each inoculated into 1 mL of LB. A sterile cotton swab was used to spread the culture over a LB plate. Liofilchem R MTS E-tests were placed separately onto the plates after inoculation for cefepime (A), ciprofloxacin (B) and tetracycline (C). When compared to wild-type and pmtA:pmtA PAO1 strains, the pmtA mutant was significantly more susceptible to cefepime and ciprofloxacin. One-way ANOVA analysis was performed (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
with pmtA:pmtA and wild-type was less than 20%. These data indicate a role for PmtA in the virulence of P. aeruginosa.

DISCUSSION
Pyocyanin and biofilm formation are powerful virulence factors for P. aeruginosa, especially in invasive lung infections. Pyocyanin is easily diffusible through host membranes and can induce a variety of harmful cellular effects including neutrophil apoptosis, alter the expression of a number of cytokines and change the redox environment leading to the persistence of harmful oxidants produced by host polymorphonuclear leukocytes (PMNs) and macrophages (Usher et al., 2002;Managò et al., 2015;Hall et al., 2016). Here, we show for the first time that a bacterial MT, PmtA, can alter the amount of pyocyanin produced by P. aeruginosa, thereby influencing biofilm formation.
Pyocyanin can influence biofilm formation in two different ways. First, it can directly accept electrons from NADH or NADPH during aerobic respiration. This process passes electrons to the final electron donor to generate reactive oxygen species (e.g., superoxide and hydrogen peroxide) (Gonçalves and Vasconcelos, 2021). The oxidative stress caused by ROS production has been shown to cause a portion of the P. aeruginosa cell population to undergo autolysis, releasing eDNA, suggesting that pyocyanin can mediate eDNA production and help promote biofilm formation (Okshevsky and Meyer, 2015;Das et al., 2016;Malhotra et al., 2019). Pyocyanin has also been shown to influence biofilm formation by directly interacting with eDNA. This binding can mediate aggregation and cell-cell interaction within biofilms, stabilizing the biofilm structure (Das et al., 2013). eDNA is an essential component of the extracellular polymeric substance produced by P. aeruginosa and has been implicated in early attachment/biofilm formation and resistance to antimicrobials (Malhotra et al., 2019). The pmtA mutant exhibited a defect in biofilm formation and/or stabilization, which is likely due to less pyocyanin availability. Interestingly, GSH restored both the pyocyanin levels and the biofilm defect in the pmtA strain. GSH neutralizes oxidative stress, mediated by pyocyanin, and P. aeruginosa gsh mutants produce less pyocyanin (Van Laar et al., 2018). Together with previous data indicating that PmtA may be upregulated after oxidant exposure, these FIGURE 8 | Wild-type PAO1 is more lethal than the pmtA mutant in a G. mellonella model. G. mellonella were injected with 5 µL of 10 3 /mL of either P. aeruginosa wild-type, pmtA and pmtA:pmtA PAO1 strains or 10 mM of MgSO 4 (control). G. mellonella survival was observed for 48 h for all four groups, and percent survival was calculated. pmtA had a higher survival percentage than PAO1 and pmtA:pmtA. The data are presented as the mean and standard deviation of the mean of three independent experiments (n = 10/group).
results suggest a role for PmtA in relieving oxidative stress. Alternatively, PmtA may interact with P. aeruginosa-derived GSH to promote pyocyanin levels. Future studies will focus on investigating potential PmtA interactions.
We also observed a decrease in the expression of phzM, a gene that encodes for methyl-transferase. This enzyme helps convert phenazine-1-carboxylic acid to 5-methylphenazine-1carboxylic acid betaine allowing for the synthesis of pyocyanin. The low transcript levels of phzM detected in the pmtA mutant compared to the wild-type strain correspond with the pyocyanin production phenotypes observed for these strains. Interestingly, PhzM expression is QS mediated, and QS has been shown to enhance the stress response in P. aeruginosa, allowing for less social cheating to occur in the population (García-Contreras et al., 2014;Chen et al., 2019). Although we did not observe a defect in AHL production within the pmtA mutant, this does not rule out that PmtA could interact with one of the four QS systems. The mechanism by which PmtA influences phzM expression in early stationary cultures remains to be understood.
Low molecular-weight thiols are essential to the detoxification of oxidative stressors, and our data suggests that PmtA may play a role in relieving oxidative stress. Interestingly, the pmtA mutant showed no growth defect when cultured in the presence of inhibitory levels of oxidative or heavy metal stressors, indicating that PmtA is not essential under these conditions. A potential reason for this lack of sensitivity may be the upregulation of yet to be identified proteins and enzymes involved in protection against these stresses that could compensate for the reduction in PmtA expression. In mycobacterial thiol mutants, defective for mycothiol and ergothioneine production, organic hydroperoxide resistance protein (Ohr) levels are upregulated to promote oxidative stress protection (Ta et al., 2011;Emani et al., 2013;Singh et al., 2016). Like mycobacteria, the absence of PmtA in the pmtA strain may lead to upregulation of essential proteins for protection against oxidative stress. However, if this were the case, the phenotypic restoration that occurred upon the addition of GSH may not have been observed.
Alternatively, as P. aeruginosa has many systems dedicated to the regulation of oxidative and heavy metal stress (i.e., a catalase, siderophores, efflux pumps and metal reduction), these systems may compensate for the lack of PmtA in the pmtA mutant, resulting in no defect being observed under the assayed growth conditions. Previous studies have shown similar results where the absence of GSH had no impact on sensitivity to hydrogen peroxide or the organic peroxide cumene hydroperoxide (CHP) (Van Laar et al., 2018). Some cyanobacteria also regulate MT production by SmtB, a zinc-dependent repressor. The PmtA operon does not encode metal-responsive regulators (Morby et al., 1993;Blindauer, 2008). Instead, the predicted operon includes two genes, one with unknown function and putatively short-chain dehydrogenase (Mao et al., 2009). In addition, Habjanič et al. (2020) noted that PflQ2 MT expression was elevated during stationary phase with or without the addition of metals in P. fluorescens Q2-87. The addition of metals at exponential phase did not induce transcription of MT, and increasing metal concentrations resulted in a decline in growth. At both exponential and stationary phases, we also did not observe any difference in growth among the assayed strains, supporting the conclusion that PmtA is not regulated by heavy metals.
Antibiotic resistance is a worldwide health crisis and the WHO has recently added carbapenem-resistant P. aeruginosa to the list of pathogens for which there is a critical need for new antibiotic treatments (Pang et al., 2019). Prolonged persistence of oxidants at low levels may be advantageous for antibiotic protection in bacteria. P. aeruginosa expresses efflux pumps that serve to expel antibiotics from the bacterial cell and thus protect the cell. An oxidant-sensitive negative regulator, MexR, controls the expression of this efflux pump (Poole et al., 1996). Oxidants cause a conformational change in MexR, which drives efflux pump transcription (Chen et al., 2008). Therefore, a decrease in extracellular oxidant production could limit the effective expression of the efflux pump and make P. aeruginosa more susceptible to antibiotics. In the present study, we showed that the pmtA mutant is more susceptible to two of the conventional antibiotics used for treatments of P. aeruginosa in cystic fibrosis patients. These results suggest that PmtA expression could make P. aeruginosa more resistant to antibiotics.
Interestingly, the effects of tetracycline were unchanged in all three strains. The difference in sensitivity could be due to the difference in antibiotic resistance classifications (i.e., intrinsic, acquired, and adaptive). Resistance to tetracycline would fall under an intrinsic classification and would include outer membrane permeability, expression efflux pumps or production of antibiotic-inactivating enzyme, whereas the development of resistance to cefepime and ciprofloxacin would fall under adaptive resistance (Reygaert, 2018). One hypothesis is that this tolerance is linked to biofilm protection, which limits the access of the antibiotic. Since the pmtA mutant has a weakened ability to form biofilms, this phenomenon could explain the increased sensitivity to these two therapeutics. Alternatively, pyocyanin levels have been linked to MDR P. aeruginosa strains, with higher levels being found in MDR vs. non-MDR strains . This suggests that the pmtA mutant's lower levels of pyocyanin could convey sensitivity, possibly due to a decrease in oxidant production.
The substantial decrease in pyocyanin levels and biofilm formation in the PmtA-deficient pmtA strains suggests that this strain is less able to influence the host immune response and thus would be less able to establish a persistent infection. The results reported in the present study suggest that a deficiency in PmtA expression results in a significant change to P. aeruginosa virulence. We used G. mellonella larvae to assess the importance of PmtA in the innate immune response as a virulence factor. Our results showed the pmtA mutant was 90% less virulent than wild-type PAO1. The virulence was restored in the complemented strain pmtA:pmtA, indicating that PmtA is an important protein for establishing infection in a host. Taken together, these results indicate that PmtA can affect P. aeruginosa virulence by influencing pyocyanin expression.
It appears likely that therapeutic manipulation of PmtA during infection could decrease pyocyanin production and reduce biofilm formation. This disruption of PmtA may be an easier therapeutic target than interfering with the several genes involved in pyocyanin expression production. This work also demonstrates the complex regulation of pyocyanin, which is governed in part by PmtA expression. Since pyocyanin expression and biofilm formation could be rescued with an exogenous antioxidant addition, PmtA likely controls the transcription of the genes involved in pyocyanin expression by altering the redox environment. A change in the redox environment could also affect the expression of other proteins involved in P. aeruginosa virulence. The results of the present study show that PmtA affects the expression of virulence factors involved in establishing P. aeruginosa infection. Thus, targeting PmtA may represent a new, innovative option in treating the human pathogen P. aeruginosa.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
AT planned, performed the deletion and complementation of pmtA, bacterial growth curves, pyocyanin extraction, qPCR, biofilm assay, waxworm infection, and wrote the manuscript. KP planned, performed bacterial growth curves, pyocyanin extraction, biofilm assay, waxworm infections, and wrote the manuscript. ML planned, supervised the experiments, and helped wrote the manuscript. MM-M planned, performed acyl homoserine lactone-dependent QS assay, bacterial growth curves, MIC determination, and waxworm infections, and helped wrote the manuscript. CM performed bacterial growth curves, pyocyanin extractions, and GSH recovery experiments. JM planned deletion, complementation of mutants, and edit manuscript. JG planned and discussed the experiments. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We want to thank Dr. Schweizer for providing the pEX18Gm plasmid.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2021.789765/full#supplementary-material Supplementary Figure S1 | Schematic representation of P. aeruginosa pmtA knock-out strain construction. Schematic indicates the generation of the pmtA mutant.
Supplementary Figure S3 | P. aeruginosa strains PAO1, pmtA, and pmtA:pmtA grow at similar rates. Single colonies of the P. aeruginosa strains were each grown overnight in 5 mL of LB media at 37 • C with vigorous shaking. These cultures were transferred to (A). 50 mL LB media at a dilution of 1:100 and grown at 37 • C with vigorous shaking. Growth was measured at 2, 4, 6, 24, and 30 h by removing 250 µL of culture in triplicate and transferring to a NUNC 96 well plate. The plate was measured using a Spectramax microplate reader. (B) 1 mL of 1:100 diluted cultures were transferred to a NUNC 96 well plate in triplicate. Growth was monitored over 15 h at 37 • C with 5 s shaking every 5 min in a Spectramax microplate reader at OD 600 . (C) Cultures were grown in M9 salts media in a 24 well plate and monitored over 15 h at 37 • C with 5 s shaking every 1 h in a Spectramax microplate reader at OD 600 . The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
Supplementary Figure S4 | PmtA is not required for survival in oxidative stress conditions. Overnight cultures were diluted at 1:100 and grown for 2 h. Cultures were adjusted to 0.08 OD at OD 600 . 600 µL of culture in triplicate were put in a 24-well plate containing either LB (A) or M9 salt media (B). Hydrogen peroxide to a final concentration of 10 mM or water was added to the wells. (A) Growth was measured at OD 600 every hour with shaking before and after measurement in a Spectramax microplate reader. The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
Supplementary Figure S5 | PmtA is not required for survival in heavy metal stress conditions. Overnight cultures were diluted at 1:100 and grown for 2 h in LB or M9 salt media. Cultures were adjusted to 0.08 OD at OD 600 . 600 µL of culture in triplicate were put in a 24-well plate. Zinc chloride (A,B) to a final concentration of 200 µM, or cadmium chloride (C,D) to a final concentration of 100 µM. Growth was measured at OD 600 every hour with shaking before and after measurement in a Spectramax microplate reader. The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
Supplementary Figure S6 | PmtA does not play a role in iron uptake. Wild-type PAO1, pmtA, and pmtA:pmtA cultures were grown overnight in M9 salt media, diluted at 1:100 and grown in M9 salt media until they reached an OD 600 of 0.08. 600 µL of culture in triplicate were put in a 24-well plate. M9 salt media (orange), EDDA to a final concentration of 150 µM (blue), EDDA + FeCl 2 (black) to a final concentration of 150 µM and 50 µg, or EDDA + Transferrin (purple) to a final concentration of 150 µM and 200 µg. Growth was measured at OD 600 every hour with shaking before and after measurement in a Spectramax microplate reader. The circles indicate PAO1, the squares indicate pmtA, and the triangles indicate pmtA:pmtA. The data are presented as the average of three biological replicates (± standard error of the mean) and are representative of three separate experiments.
Supplementary Table S1 | Bacterial strains and plasmids used in the present study.
Supplementary Table S2 | Primers used in the present study.