Investigation of Zur-regulated metal transport systems reveals an 3 unexpected role of pyochelin in zinc homeostasis

28 Limiting the availability of transition metals at infection sites serves as a critical defense mechanism 29 employed by the innate immune system to combat microbial infections. Pseudomonas aeruginosa 30 exhibits a remarkable ability to thrive in zinc-deficient environments, which is facilitated by intricate 31 cellular responses governed by numerous genes regulated by the zinc-responsive transcription factor 32 Zur. Many of these genes have unknown functions, including those within the predicted PA2911-33 PA2914 and PA4063-PA4066 operons. A bioinformatic analysis revealed that PA2911-PA2914 34 comprises a TonB-dependent outer membrane receptor and an inner membrane ABC-permease 35 responsible for importing metal-chelating molecules, whereas PA4063-PA4066 contains genes 36 encoding a MacB transporter, likely involved in the export of large molecules. Molecular genetics 37 and biochemical experiments, feeding assays, and intracellular metal content measurements 38 demonstrated that PA2911-PA2914 and PA4063-PA4066 are engaged in the import and export of the 39 pyochelin-cobalt complex, respectively. Notably, cobalt can reduce zinc demand and promote the 40 growth of P. aeruginosa strains unable to import zinc, highlighting pyochelin-mediated cobalt import 41 as a novel bacterial strategy to counteract zinc deficiency. These results unveil an unexpected role for 42 pyochelin in zinc homeostasis and challenge the traditional view of this metallophore exclusively as 43 an iron transporter. 44 45


Introduction
Zinc (Zn) is an essential micronutrient for all living organisms, serving as a structural or catalytic cofactor in numerous proteins involved in fundamental biological processes.In prokaryotes, Znbinding proteins constitute approximately 6% of the proteome, including proteins involved in central metabolism, DNA replication and repair, antibiotic resistance, and virulence pathways (1,2).The intracellular concentration of this ion is finely regulated in cells to prevent problems due to imbalances.Indeed, Zn deficiency can lead to the loss of functionality of Zn-dependent proteins, whereas excess Zn can have toxic effects, including aberrant interaction with proteins and cellular components and competition with other relevant metal ions (3,4).
The importance of Zn is underscored by the concept of "nutritional immunity", where vertebrates modulate the bioavailability of Zn and other transition metals at the host-pathogen interface to combat bacterial infections (5).As part of this defense mechanism, macrophages can accumulate Zn in phagosomes to poison the internalized pathogens with high concentrations of the metal (6,7).
Conversely, serum or mucosal surfaces deplete Zn to hinder microbial growth during infections.This depletion occurs through metal redistribution among tissues and the action of metal-chelating proteins, such as the calprotectin released by neutrophils (8,9) Pathogens have evolved adaptive mechanisms to acquire and maintain sufficient Zn levels to circumvent nutritional immunity.For instance, the high-affinity Zn-import system ZnuABC, a transporter of the ABC family widely conserved across many bacterial species, plays a crucial role in counteracting Zn deficiency by actively translocating Zn into the bacterial cytoplasm (2,10).Studies carried out in many bacteria, including Salmonella enterica, uropathogenic Escherichia coli (UPEC), Brucella abortus, and Yersinia pestis, have demonstrated a remarkable loss of pathogenicity when this system is inactivated (11)(12)(13)(14).
Certain pathogenic bacteria employ additional Zn acquisition strategies, including synthesizing and releasing low molecular weight metal-binding molecules (metallophores) that act as extracellular metal chelators, facilitating selective Zn import.Zn-specific metal-binding metallophores, often .CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted January 8, 2024.; https://doi.org/10.1101/2024.01.07.574578 doi: bioRxiv preprint called zincophores, have been identified in Staphylococcus aureus (15), Pseudomonas aeruginosa (16), and a few other bacterial species (17) and have been proven to contribute significantly to the ability of these bacteria to cause infection (18,19).As an example, in P. aeruginosa the genes located in the zrmABCD operon are involved in the synthesis and selective transport of pseudopaline, a metallophore mediating Zn import.This operon includes the genes encoding for the TonB-dependent transporter ZrmA, the nicotianamine synthase ZrmB, the opine dehydrogenase ZrmC and the inner membrane exporter ZrmD (16).
P. aeruginosa is a Gram-negative opportunistic pathogen responsible for infections that can be particularly threatening for immunocompromised individuals (20).In fact, its metabolic versatility, intrinsic resistance to many antibiotics, and ability to form biofilm, favor its rapid adaptation in the host environment, where it can give rise to both acute and chronic infections that are very difficult to treat.P. aeruginosa is particularly adept at responding to Zn fluctuation.It can withstand Zn deficiency conditions through the high-affinity Zn import system ZnuABC and the zincophore pseudopaline, which is synthesized and released in the Zn-limited environments characteristic of host tissues during infections (19).The functionality of these Zn import systems is crucial for P. aeruginosa to express a wide range of virulence features under conditions of Zn deficiency, allowing it to cause lung and systemic infections (19,21,22).Reduced Zn uptake affects the ability of P. aeruginosa to release extracellular Zn-dependent proteases, impairs swarming and swimming motility, hinders the synthesis of the exopolysaccharide alginate and biofilm formation, and reduces the release of the siderophore pyoverdine (22).Additionally, Zn-binding metallophores can interfere with the ability of calprotectin to sequester Zn, enhancing P.aeruginosa resistance to the nutritional immunity of the host (23).Moreover, P. aeruginosa produces and releases several metallophores, most of which have been characterized for their ability to deliver iron (Fe) to the cell (24).Even if these molecules are primarily involved in Fe transport, it should be remembered that they can strongly chelate other metals, as previously documented for the siderophore pyochelin (25,26).
. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted January 8, 2024.; https://doi.org/10.1101/2024.01.07.574578 doi: bioRxiv preprint P. aeruginosa response to Zn deficiency is transcriptionally regulated by the Zn-uptake regulator protein Zur, which senses intracellular Zn levels and modulates the expression of its target genes (27,28).Among the genes regulated by Zur, there are those included in the znuABC and zmrABCD operons, responsible for the production of the previously mentioned Zn import systems Numerous genome-wide gene expression studies have identified Zur-regulated genes between the most highly expressed genes in P. aeruginosa recovered from infected tissues or cultured under host-mimicking conditions.These genes are overexpressed in various infection scenarios, underscoring their importance for the ability of P. aeruginosa to colonize the host environment (19,(21)(22)(23)(24)(25)(26).
Notably, the P. aeruginosa Zur regulon encompasses a larger number of genes than other bacteria and includes genes unique to this organism and whose role is not yet known (19,29).Given the high expression of many of these orphan genes during infection, understanding their role may foster our understanding of the interaction between P. aeruginosa and its host.This study explores the role of two uncharacterized Zur-regulated gene clusters, PA2911-2914 and PA4063-4066, annotated as operons encoding putative metal import systems.The reported results demonstrate that these operons encode transport systems responsible for the import and export of the pyochelin-cobalt complex.Such results indicate that P. aeruginosa can adapt to Zn deficiency by substituting this metal with cobalt and reveal an unexpected role for pyochelin in Zn homeostasis, highlighting its multifunctional role beyond Fe transport.

Results
The PA2911-2914 and PA4063-4066 operons are regulated by Zn availability.
The experiments reported in this paper were performed with the P. aeruginosa strain PA14.However, for ease of comparison with the extensive body of literature reporting data on these predicted operons, we will adopt the nomenclature of the reference strain PAO1.Consequently, we will refer to the operon PA14_26420-PA14_26360 as PA2911-2914 and to the operon PA14_11320-PA14_11280 as PA4063-PA4066 (Fig 1).Zur-dependent regulation of these operons.Under Zn-deficient conditions, the expression of PA4063 is comparable in wild-type PA14 and in the znuAzrmB double mutant strain.Conversely, the expression of PA2911 significantly increases in the znuAzrmB background, where the intracellular concentration of Zn is lower than in the wild type (Fig. 2A, znuAzrmB bar).This observation implies a graded response of different Zur-regulated genes depending on the intracellular Zn availability.
To investigate the role of PA2911-PA2914 and PA4063-PA4066 in Zn homeostasis, we deleted either PA2914 or PA4065, the predicted inner membrane permeases of each operon, in both the PA14 wildtype and the znuAzrmB double mutant backgrounds.The growth curves of the resulting mutants (PA2914, PA4065, PA2914znuAzrmB, and PA4065znuAzrmB) were compared to their respective parent strains (i.e., wild-type and znuAzrmB) under Zn-restricted conditions.Figure 2B shows that neither PA2914 nor PA4065 inactivation impairs bacterial growth.However, when either PA2914 or PA4065 mutations are introduced into the znuAzrmB mutant strain, which lacks crucial Zn uptake systems, a marked decrease in bacterial growth is observed under low Zn availability conditions compared to a znuAzrmB mutant strain.The simultaneous deletion of PA4065 and PA2914 in the znuAzrmB background (resulting in the mutant strain PA2914PA4065znuAzrmB) does not cause any growth difference compared to each triple mutant (S1 Fig. ).
Next, since zrmA expression is significantly influenced by intracellular variations in Zn concentration (19), we employed the Zn-responsive zrmA promoter to assess whether the inactivation of PA2914 or PA4065 could alter the intracellular Zn status in bacteria grown in Zn-limited conditions.For this purpose, plasmid pzrmApTZ110 (19) was introduced into wild-type PA14 and a panel of mutant strains with different combinations of mutations.The activity of the zrmA promoter was assessed in cultures grown in Zn-restricted conditions.The results presented in Figure 2C confirm that zrmA promoter activity is higher in the znuAzrmB strain than in the wild-type strain (19).Promoter activity increases even further in the PA4065znuAzrmB mutant, suggesting that the absence of PA4065 intensifies the intracellular demand for Zn.In contrast, there are no differences in zrmA expression in the PA2914znuAzrmB and znuAzrmB strains.Intriguingly, the simultaneous deletion of PA2914 and PA4065 in a znuAzrmB background restores the zrmA promoter activity to the same level found in the znuAzrmB strain.
In summary, the results just described suggest that both PA2911-PA2914 and PA4063-PA4066 gene clusters are negatively regulated by Zn and contribute to the ability of P. aeruginosa to adapt to a Znlimited environment.While the observed impact of simultaneous inactivation of both operons on the modulation of the Zur-responsive gene zrmA suggests a functional relationship between the two predicted operons, PA4065 knock-out induces a stronger response of the zrmA promoter compared to the PA2914 knock-out.

PA4064 and PA4065 encode for a MacB-type exporter.
A structural bioinformatics analysis was conducted to predict the functions of the proteins encoded by the PA2911-2914 and PA4063-PA4066 operons.Using the SWISS-MODEL web server, it was predicted that PA2911 serves as a TonB-dependent outer membrane receptor.At the same time, the PA2912-PA2914 genes collectively encode an ABC family import system.Notably, all four components of this operon exhibit significant similarity to proteins involved in siderophore-mediated Fe uptake.
In addition, AlphaFold 2 was employed to predict the three-dimensional structures of PA4064 and PA4065.The analysis revealed that the two proteins form a complex with a fold characteristic of a MacB transporter, which is a class of ABC transporter that collaborates with other adaptor proteins to facilitate the efflux of antibiotics, toxins, or siderophores out of the bacterial cell (31).MacB transporters generally consist of two interacting MacB monomers, each containing four transmembrane helices, an N-terminal nucleotide-binding (NBD) domain, and a long periplasmic domain between TM1 and TM2.In this case, the putative MacB transporter predicted by Alphafold

Pyochelin production is downregulated in the znuAzrmB mutant strain.
Differences in the pigmentation of liquid cultures between the wild-type and the znuAzrmB strain of P. aeruginosa PA14 grown overnight under Zn-limiting conditions (VBMM) were previously reported (22).Notably, the wild-type strain showed a greenish tint, while the znuAzrmB cultures were characterized by a pale blue color.This difference was correlated with a decrease in pyoverdine (PVD) production (22).In this study, we observed that the inactivation of either PA2914 or PA4065 in the znuAzrmB strain caused the cultures to regain a color very similar to that of the wild-type PA14 Besides PVD, which is produced in low amounts by the PA14 strain in these conditions, P. aeruginosa secretes other pigments that absorb light in the UV-visible spectrum.Therefore, we decided to investigate whether these phenotypic differences could be attributed to changes in the abundance of one of these metabolites by analyzing the aqueous and ethyl acetate fractions obtained from cell-free supernatant.Fluorimetric studies on the aqueous fraction confirmed the reduction of PVD secretion in the znuAzrmB mutant previously observed in the znuAzrmA mutant strain (22).Even if differences in PVD production can contribute to the different colors of bacterial cultures, the ethyl acetate fractions, which contain pigments less hydrophilic than PVD, exhibited much more marked differences.
Albeit with small quantitative differences, the UV-visible spectra of the ethyl acetate fractions from the wild-type, PA2911znuAzrmB, and PA4065znuAzrmB strains all displayed three major peaks around 250, 315, and 370 nm (Fig 4A).In contrast, the znuAzrmB sample showed only a weak peak around 315 nm.Since the absorption spectrum of purified apo-PCH is characterized by two major peaks at 248 and 313 nm (32), we hypothesized that the color changes among the different supernatants could be largely due to differences in PCH secretion.The peak at 370 nm is likely due to residual pyocyanin contamination (33)   in PCH uptake (ftpA and fptX), regulation (pchR), and synthesis (pchD and pchE), in the znuAzrmB mutant grown in E-VBMM, relative to the PA14 wild type.Data are mean values ± S.D. of triplicates, and statistical analyses were performed by Two-way ANOVA and Sidak's multiple comparison test.Asterisks indicate statistically significant differences between the znuAzrmB strain and wild-type, grown in the same conditions (**p<0.005;***p<0.0005;****p < 0.0001; ns, not significant).

Zn
The results discussed in the previous section demonstrate a significant decrease in PCH production in the znuAzrmB mutant that is largely restored when the mutations in znuA and zrmB are combined with the deletion of PA2914 or PA4065 (S3 Fig. ).Furthermore, based on activation of the of zrmA promoter, it was observed that the intracellular requirement for Zn is greater in the PA4065znuAzrmB triple mutant compared to the znuAzrmB double mutant.However, the further deletion of PA2914 restores expression of zrmA to levels observed in the double mutant (see Figure 2C).These findings led us to hypothesize that PA2911-PA2914 and PA4063-PA4066 operons are involved in the transport of PCH in and out of the cell.This mechanism could be related to bacterial adaptation when facing severe Zn limitation.
To explore this hypothesis, we analyzed the transcriptional activity of zrmA in the mutant strains znuAzrmB, PA2914znuAzrmB, and PA4065znuAzrmB compared with each isogenic pchE mutant (znuAzrmBpchE, PA2914znuAzrmBpchE, and PA4065znuAzrmBpchE), grown in E-VBMM.The pchE strains cannot produce mature PCH due to the inactivation of one of the enzymes involved in the biosynthetic process.Figure 5A shows that the zrmA promoter is expressed at higher levels in the znuAzrmBpchE strain than in the znuAzrmB strain, suggesting that the absence of PCH reduces the intracellular Zn content.No changes in transcription were observed when the pchE and PA2914 mutations were combined, consistent with the hypothesis that the PA2911-2914 operon encodes a .CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted January 8, 2024.; https://doi.org/10.1101/2024.01.07.574578 doi: bioRxiv preprint PCH import system.Remarkably, zrmA transcription is restored to levels seen in the znuAzrmB strain when the deletion in pchE is introduced in the PA4065znuAzrmB mutant strain, indicating a role for PCH in the high activity of the zrmA promoter in the triple mutant.No changes in transcription were observed when the pchE and PA2914 mutations were combined, suggesting that the PA2911-2914 encodes a PCH import system.
The impact of PCH on the Zn intracellular status was then analyzed by assessing the activity of the zrmA promoter in PA2914znuAzrmB, PA2914znuAzrmBpchE, PA4065znuAzrmB, and PA4065znuAzrmBpchE strains, grown in VBMM supplemented with the ethyl acetate fraction from wild-type or pchE strain.As shown in Figure 5B, no significant differences were observed among the PA2914 defective strains, indicating that the disruption of this importer has no impact on intracellular Zn availability, either in the presence or absence of PCH in the growth medium.In contrast, feeding with a PCH-containing supernatant induces the zrmA promoter in strains lacking PA4065 (Figure 5B).These results suggest that the increased expression of zrmA in the PA4065znuAzrmB mutant may be attributed to the intracellular accumulation of PCH, causing dysregulation of Zn homeostasis, and that PA4065 plays a role in the export of PCH.

PA2914 and PA4065 are involved in PCH trafficking
To test the hypothesis that PA4065 is involved in PCH export, we used a luminescent biosensor (34) to measure the concentration of PCH in cell-free supernatants and cell lysates from the wild-type, znuAzrmB, PA4065 and PA4065znuAzrmB strains (Fig 6).This analysis not only confirmed that PCH production is below the detection limit in the znuAzrmB strain but also revealed that the deletion of PA4065 significantly enhances the intracellular accumulation of PCH, particularly in the PA4065znuAzrmB triple mutant.PCH secretion was comparable among the other strains, with a slight yet significant increase observed in the PA4065znuAzrmB supernatant.The ability of mutants lacking PA4065 to export PCH and the increase of extracellular PCH in the triple mutant, which appears to be correlated with the high concentration of intracellular PCH in this strain, confirms the existence of PCH export mechanisms independent from the PA4064-PA4065 MacB exporter identified in this study.The influence of PCH on intracellular Zn homeostasis was evaluated by supplementing the growth medium with purified apo-PCH and analyzing the zrmA promoter activity in different mutant strains ( Fig 7).Exogenous PCH significantly increases the Zn demand in all strains except the one lacking PA2914.This differential response of the various strains to incubation with PCH seems to exclude that the increase in zrmA expression is due to a chelating effect of PCH, whose ability to bind Zn is well known (26,35).Instead, this observation aligns with the hypothesis that PA2914 plays a role in PCH uptake.The inactivation of PA4065 or PA2914 has an impact on intracellular cobalt content.
To investigate the hypothesis that PA2911-PA2914 and PA4063-PA4066 are involved in a PCHmediated mechanism of metal acquisition aimed at counteracting Zn-starvation conditions, we measured the metal content in various mutant strains grown in E-VBMM supplemented with trace amounts of transition metals.Consistent with previous observations, the Zn content of the znuAzrmB mutant is significantly lower than that determined for both the wild-type and the znuA mutant strains, confirming the crucial role of pseudopaline in adapting to Zn-deficiency (19).The introduction of PA2914, PA4065 or pchE deletions into the znuAzrmB strain did not further decrease cellular Zn content (Fig 8 , left panel).This suggests that neither PCH nor PA4065 or PA2914 is directly involved in Zn uptake under conditions of metal restriction.In addition, no significant differences were observed in these strains with respect to metals such as copper (Cu), nickel (Ni), or manganese (Mn).
While changes in Fe content were moderate, strains lacking pchE exhibited a trend towards decreased Fe content (S3 Table ).In contrast, substantial differences were observed in cobalt (Co) content (Fig  hash signs indicate pairwise statistically significant differences ( #### p < 0.0001; ns = not significant).

The absence of PCH alleviates the cobalt-dependent growth impairment.
The PA14 wild-type strain is sensitive to Co, which causes a growth impairment even at relatively low concentrations.In contrast, we have shown that Co promotes the growth of the znuAzrmB mutant, indicating that this metal can at least partially compensate for low Zn availability (19) where PCH biosynthesis is abolished, displays no growth defect when grown in the presence of Co.

Discussion
The ability to efficiently acquire Zn in environments where this metal is poorly available plays a crucial role in the interaction of P. aeruginosa with its hosts.This is demonstrated by the impaired expression of several virulence features and the marked loss of virulence of strains with reduced Zn acquisition ability (19,36).P. aeruginosa exhibits robust responses to Zn deficiency, as evidenced by its efficient proliferation in Zn-poor media and resistance to the antimicrobial activity of the Znsequestering protein calprotectin, even in the absence of a functional ZnuABC transporter (21,28).
The identification of an extracellular metal capture mechanism based on the pseudopaline metallophore produced by the ZrmABCD system has provided evidence that P. aeruginosa uses additional systems to obtain Zn (16,19).However, our understanding of P. aeruginosa adaptations to Zn deficiency is still limited.Notably, the Zur operon in this species includes over 30 genes, many of which are yet to be characterized, and some of which are not found in closely related Pseudomonas species (29).For instance, the zrmABCD operon, involved in pseudopaline synthesis and transport, is highly conserved in all P. aeruginosa strains, including clinical isolates, but is not found in other closely related species colonizing soil, water, and plants such as P. fluorescens, P. putida, P. stutzeri and P. syringae.This suggests that pseudopaline could be particularly important for promoting Zn recruitment within animal hosts.
To shed light on the function of Zur-regulated genes of unknown function, this study aimed to investigate two previously uncharacterized Zur-regulated operons, PA2911-2914 and PA4063-4066, both of which include genes encoding putative metal transporters (Fig. 1).
The results presented in Fig. 2 confirm that both operons are regulated by Zn.Mutants lacking PA2911 or PA4065 show similar growth to the wild-type strain in a Zn-poor medium.However, when these mutations were introduced into a strain lacking ZnuA and ZrmB, a significant reduction in growth was observed, highlighting the contribution of these two operons to adaptation under conditions of Zn deficiency, especially in bacteria strongly defective in Zn acquisition.
However, the results reported in Fig. 2C raise questions regarding the mechanisms by which these operons contribute to adaptation under conditions of Zn deficiency.Transcription arising from the zrmA promoter, that is sensitive to changes in intracellular Zn concentration, was assessed introducing the Zur-responsive zrmA::lacZ promoter fusion vector in different mutants.It was observed that zrmA expression is significantly higher in the PA4065znuAzrmA triple mutant than in the znuAzrmA double mutant.However, the expression of zrmA in the PA2911PA4065znuAzrmA quadruple mutant was indistinguishable from that observed in the znuAzrmB mutant, suggesting a functional relationship and compensation between the systems encoded by the two operons.
To find an interpretation of these results, we performed a bioinformatic analysis on the proteins encoded by the PA2911-2914 and PA4063-PA4065 operons.The PA2911-2914 operon encodes a TonB-dependent outer membrane receptor (PA2911) and the components of a classical importer of the ABC family (PA2912-PA2914).These proteins show similarities with other transporters involved in the entry of siderophores or vitamin B12 into the cell, leading to the hypothesis that they constitute a system capable of mediating the transport of a metal bound to a metallophore from outside the cell to the cytoplasm.More unexpected were results deriving from the analysis of the PA4063-4066 operon.Previous studies have annotated this system as an ABC transporter involved in Zn uptake (29,37).However, a protein prediction analysis carried out with Alphafold revealed that the PA4064 and PA4065 proteins are subunits of an exporter of the MacB family.MacB proteins represent an atypical family of ABC transporter described in many Gram-negative bacterial species, including E. coli.These transporters typically bind a MacA component and the inner membrane protein TolC to form a tripartite efflux pump.The tripartite complex MacA-MacB-TolC is a transmembrane machinery that extends both on the plasma membrane and on the outer membrane and actively extrudes large molecules, including macrolide antibiotics, virulence factors (e.g., pyoverdine in the case of P. aeruginosa), peptides and precursors of the cell envelope (38)(39)(40)(41).The main function of the MacB transmembrane domain is to transmit conformational changes from one side of the membrane to the other, with a process defined mechanotransmission (38).While previously characterized MacB transporters were encoded by a single polypeptide chain, this P. aeruginosa MacB is formed by the association of two independent polypeptides, with PA4064 forming the ATP binding domain and PA4065 encoding the membrane channel.Despite this difference in gene organization, the predicted structure of PA4064/PA4065 perfectly overlaps the structure of known MacB proteins.Although further studies are needed to clarify the export mechanism of this unique MacB transporter, it is important to note that this organization is conserved in other bacterial species.For example, a gene cluster analogous to PA4063-PA4066 is present in Vibrionaceae and other bacteria, all under the control of Zur (42).The other two operon members, namely PA4063 and PA4066, encode for soluble periplasmic proteins of known structure but still unclear function.PA4063 is a metal-binding protein able to bind Zn or Co ions at relatively low affinity, proposed to play a role as a periplasmic metal chaperone (30).In Vibrio cholerae, its homologous protein, ZrgA, is required for the proper function of the whole transport system (43).
The crystal structure of PA4066 has revealed that this protein may assemble in a tetrameric structure with no evident ability to bind Zn.Although the function of PA4063 and PA4066 remains undisclosed, the observation that even in other bacteria the genes encoding their homologs are invariably associated with those for proteins like PA4064 and PA4065 corroborates their involvement in supporting the export functions of the MacB transporter.
The identification of PA4063-4066 as an export system led to the hypothesis that the results shown in Fig. 2C could be rationalized assuming that the PA2911-2914 uptake system is responsible for the internalization of a metallophore which must then be eliminated from the bacterial cytoplasm through the action of the MacB exporter coded by PA4063-4066.The intracellular accumulation of metalbinding molecules can be harmful to cells due to their ability to interfere with metal binding to proteins, which can cause a condition of apparent metal deficiency.For example, it has been shown that the intracellular accumulation of staphylopine (a molecule structurally related to pseudopaline) is toxic to Staphylococcus aureus (44,45).In line with this, it has been demonstrated that disruption of either zrmD or PA4063 caused a dramatic loss of growth ability in Zn-devoid airway mucus secretion (46).
The next challenge was to identify the substrate imported by PA2911-2914.It could have been a still unknown metallophore, perhaps produced specifically in conditions of Zn deficiency, or an already known molecule with other associated functions.The results in Fig. 4 showed a significant reduction in the release of PCH in the znuAzrmB mutant, stimulating further investigations of the potential involvement of this metal chelator in the response to Zn deficiency.PCH is well known to promote microbial growth by solubilizing ferric iron and accelerating metal transport within the cell (47).
However, in vitro, PCH can bind other metals with high affinity, including Ag + , Co 2+ , Cu 2+ , Ni 2+ , Pb 2+, and Zn 2+ (48).Moreover, Brandel et al. showed that that PCH strongly chelates divalent metals such as Zn (pZn = 11.8 at pH 7.4) forming 1: 2 (M2 + /PCH) complexes (26).While the ability of PCH to bind Zn is remarkable, it does not mediate its import into cells.This is due to the transport selectivity of the FptA receptor, which efficiently mediates the uptake of the Fe-PCH complex, and, to a lesser extent, of complexes with Ni, Co, and gallium, but not those involving other metals (48,49).
Interestingly, the ability of FptA to mediate entry of the PCH-Co complex affects the production of PCH (50).It is also worth mentioning some recent studies reporting that in Yersinia pestis, the yersiniabactin siderophore, structurally very similar to PCH, binds Zn 2+ and transports it to the cell via the transporter YbtX (13,51).Likewise, the use of yesiniabactin as a zincophore has been documented in Escherichia coli (52).
These findings led to the hypothesis that PA2911-2914 codes for an alternative PCH import system, while the MacB transporter encoded by PA4064/PA4065 serves as an export pump for PCH.Spectrophotometric analyses of bacterial supernatants (Fig. 4), feeding assays and experiments with purified PCH (Fig. 5 and Fig. 7), and measurements of PCH concentration in bacterial extracts and supernatants (Fig. 6) support this hypothesis.Moreover, an ICP-MS analysis has revealed that no relevant differences in Zn concentration were observed between the znuAzrmB strain and the PA2911znuAzrmB and PA4065znuAzrmB mutants.However, the PA4065znuAzrmB mutant exhibited .CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted January 8, 2024.; https://doi.org/10.1101/2024.01.07.574578 doi: bioRxiv preprint a disproportionate accumulation of Co, which was completely abolished in the PA4065znuAzrmBpchE mutant strain (Fig. 8).All these data converge to support the hypothesis that PA2911-2914 and PA4063-4066 gene clusters are involved, respectively, in the import and export of PCH.Specifically, the results indicate that PA2911-2914 is involved in the uptake of the PCH-Co complex while the MacB transporter is involved in the export of excess Co, possibly in complex with PCH.These observations, therefore, suggest that one of the strategies adopted by P. aeruginosa to respond to a condition of Zn deficiency is to activate a PCH-based system to supply the cell with Co.
Importantly, ICP-MS data reveal that both PCH and pseudopaline participate in the acquisition of Co.
Furthermore, the observation that PA2911 is more expressed in the znuAzrmB mutant than in the wild-type strain supports the hypothesis that this transport system becomes necessary when other routes for Zn acquisition are already defective.
The import of Co under conditions of Zn deficiency does not appear to be a causality.Co has the same atomic radius as Zn and can be incorporated in many proteins instead of Zn, functionally replacing the original metal (53).Recent studies have revealed that in Salmonella Co supplementation can rescue the growth defects caused by Zn deficiency and that Co can overcome Zn unavailability by taking the place of Zn in proteins whose function requires the presence of this metal cofactor (54).
Previous observations have revealed that, also in P. aeruginosa, Co can repress the expression of Zurdependent genes and stimulate the growth of a znuAzrmA mutant strain (19).Moreover, here we show that inactivation of the PCH biosynthetic pathway increases the demand for Zn, as assessed by the analysis of zrmA expression in the znuAzrmB and znuAzrmBpchE mutant strains (Fig 5 , Panel A).A controlled increase in Co would therefore represent a strategy implemented by P. aeruginosa to cope with Zn deficiency conditions.However, it is necessary to highlight a significant difference in the responses of Salmonella Typhimurium and P. aeruginosa to Co.While high concentrations of Co have no obvious toxic effects on Salmonella (54), 10 µM Co inhibit the growth of P. aeruginosa (19).
Results described in Figure 9 indicate that disruption of PCH synthesis ability increases resistance to Co of the PA4065znuAzrmB mutant strain, suggesting that Co toxicity could be at least partially .CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted January 8, 2024.; https://doi.org/10.1101/2024.01.07.574578 doi: bioRxiv preprint correlated with intracellular accumulation of PCH.Therefore, PCH-mediated Co uptake pathway should be regarded as a double-edged strategy to face with Zn deficiency, as limited Co intake compensates for Zn deficiency, while excess Co is toxic.It is worth noting that many Zur-regulated genes with uncharacterized functions encode proteins annotated as proteins involved in cobalamin biosynthesis or import.Some of these proteins are putative metallochaperones of the COG0523 family, that are emerging as critical players in cell adaptation to conditions of limited Zn availability (55)(56)(57).It is tempting to hypothesize that these proteins play a role in the redistribution and allocation of Zn and Co in different proteins in response to a reduced intracellular availability of Zn.
The study also raises questions about the regulation of PCH synthesis, the selectivity of receptors for PCH-metal complexes, and the reasons behind changes in the extracellular accumulation of PCH.It is evident that, apart from PA2911-2914, representative genes involved in the synthesis (pchD, pchE) and transport of PCH-Fe (fptA, fptX) are strongly repressed in the znuAzrmB mutant.In contrast, PCH synthesis is active in PA2914znuAzrmB and PA4065znuAzrmB mutants.The repression of PCH synthesis in the znuAzrmB double mutant can probably be explained based on the model of PCH synthesis regulation recently proposed by Schalk and collaborators (50).According to this model, the transcription of all PCH genes, except pchR, is stimulated by the transcriptional activator PchR in complex with PCH-Fe 3+ but is repressed by the binding of PchR to the PCH-Co complex.Under conditions of severe Zn deficiency that characterize the znuAzrmB mutant, increased expression of the PA2911-2914 operon promotes the selective uptake of the PCH-Co complex, likely resulting in inhibition of pyochelin synthesis.Although FtpA can import PCH complexed with both Fe and Co, the uptake rate of Co is 26-fold lower than that for Fe (48).If we hypothesize that PA2911-2914 provides a more effective entry route for the PCH-Co complex, this would probably explain the fact that the inhibitory mechanism is not active in the PA2914znuAzrmB mutant.It will be interesting in the future to conduct studies to characterize the binding specificity of the FptA and PA2914 receptors in greater detail.However, the reason why PCH synthesis is reactivated in the PA4065znuAzrmB mutant despite an apparent intracellular accumulation of the PCH-Co complex remains to be .CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted January 8, 2024.; https://doi.org/10.1101/2024.01.07.574578 doi: bioRxiv preprint investigated.For instance, it can be hypothesized that in the presence of high concentrations of this complex, the PchR regulator regains a transcriptionally active conformation.
Additionally, the identification of a MacB export system dedicated to PCH export requires some comment.While both the enzyme systems involved in the cytoplasmic synthesis of PCH and the transporters that mediate the entry of the PCH-Fe complex across the outer and inner membrane are well known, no protein complex involved in PCH secretion has ever been characterized (58).Given the predominantly hydrophobic nature of PCH, it has often been assumed that, once synthesized in the cytoplasm, it could permeate the membrane without a dedicated export system (59).Therefore, the MacB transporter, composed of the PA4064 and PA4065 subunits, is the first active PCH export system to be identified.This export system is under Zur regulation and is active only in conditions of Zn deficiency.Our results suggest that P. aeruginosa can export PCH even in the absence of this transporter.The mechanism for the export of newly synthesized PCH remains an open question, and membrane passage without a dedicated export system remains a valid hypothesis.Recent studies indicate that only a fraction of the PCH-Fe complex entering the periplasm via FptA is routed into the cytoplasm through FptX, while another part dissociates in the periplasm to allow free-form Fe entry through the channel formed by PchHI proteins (60).This is probably another mechanism to prevent intracellular accumulations of PCH that could be toxic to the cell.In this complex scenario, a diagram summarizing the role of the different characterized Zur-regulated metal transporters in P. aeruginosa response to Zn-deficiency is reported in Figure 10. of Zn into the periplasm are not defined).In addition to znuABCD, the operons zrmABCD (responsible for the synthesis and transport of pseudopaline), PA2911-PA2914 and PA4063-PA4066 are also negatively regulated by Zur under Zn-replete conditions and transcribed when the intracellular Zn concentration is low, a condition in which Zur is no longer properly metalated.The zincophore pseudopaline, synthesized in the cytoplasm by ZrmB and ZrmC, is transported into the periplasm by ZrmD and then exported out of the cell by the MexAB-OmpR efflux pump (61).Pseudopaline complexed to Zn or Co re-enters the periplasm via the TonB-dependent Outer Membrane Receptor ZrmA.It is currently unknown whether the pseudopaline-bound metal is released in the periplasm or transported into the cytoplasm in complex with the pseudopaline (dashed arrows through the Inner Membrane -IM).Co, which can partially compensate for conditions of severe Zn deficiency, can enter either in complex with pseudopaline or bound to PCH.The PCH-Co complex can enter the cell either from FptA, the receptor involved in the uptake of the PCH-Fe complex or through the PA2911-2914 import system.The binding of PCH-Co to PchR, the main regulator of PCH synthesis, represses the synthesis of PCH .CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted January 8, 2024.; https://doi.org/10.1101/2024.01.07.574578 doi: bioRxiv preprint and of the FptA-FptX import system (50).In contrast, the expression of PA2911-2914 is not repressed by PchR, but induced under conditions of poor Zn availability, thereby allowing the entry of PCH-Co even if fptA-fptX is repressed.To avoid potentially toxic accumulations of PCH and Co within the cell, excess PCH-Co is exported through a pump involving the MacB transporter formed by PA4064/PA4065.
Finally, the most intriguing aspect emerging from this study is a reevaluation of the paradigm regarding PCH as merely a Fe carrier.While P. aeruginosa can indeed produce pyoverdine, a siderophore with a significantly higher Fe affinity than PCH, it is now believed that under conditions of moderate Fe deficiency, PCH production is favored due to its lower energy requirements for synthesis compared to pyoverdine (62).This study unveils additional roles for PCH that extend beyond its conventional role in extracellular Fe uptake, as hinted at in a previous study (63).The findings reported here emphasize the involvement of PCH in cellular responses to Zn deficiency, shedding light on its broader role in connecting the homeostasis of Fe and Zn, two central metals in nutritional immunity.In this regard, several studies have highlighted that during infections many of the most highly expressed genes often belong to the Zur operon or are involved in the synthesis of pyochelin, suggesting that Fe and Zn are limited during infections (46,64).The identification of a role for PCH also in the adaptation of P. aeruginosa to Zn deficiency introduces new elements of complexity that suggest caution in the interpretation of transcriptomic data concerning the genes involved in the synthesis and transport of PCH.
In conclusion, our findings reveal that P. aeruginosa possesses intricate and finely tuned mechanisms for Zn import, explained by the critical role of Zn in infections.A comprehensive understanding of Zn acquisition systems can pave the way for new strategies to interfere with metal homeostasis, potentially enhancing infection control.

P. aeruginosa mutants construction
Mutant strains were obtained by the gene replacement method described by Hoang (66), following the same protocol previously used to generate znuA, zrmA, and zrmB mutants (19,21).The primers  S2. Cloning of the 5' and the 3' terminal fragments of the target genes and the gentamicin resistance cassette was performed in plasmid pEX18Tc, and the mobilization of the resulting vector from E. coli DH5α to P. aeruginosa was achieved by tri-parental mating, as already described (21).When needed, the gentamicin resistance cassette was removed from the strain using plasmid pFLP2, encoding the gene for the Flp recombinase, as previously described (21).Gene deletions or the loss of the gentamicin resistance cassette were confirmed by PCR using the primers listed in Table S2.

Analyses of bacterial growth
Overnight cultures of P. aeruginosa grown in LB were diluted 1:1000 in VBMM with EDTA 5 μM (E-VBMM).A volume of 0.2 mL of each sample was inoculated in a 96-microwell (Greiner Bio-One, Austria) and incubated at 37°C in a Sunrise™ microplate reader (Tecan, Männedorf, Switzerland).Optical density at 595 nm (OD595) was registered every hour for 24 hours.Each sample was tested in triplicate.

RNA extraction and Real Time-PCR analysis
RNA was extracted from P. aeruginosa PA14 using the RNAeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, with the addition of DNase (Qiagen) and lysozyme (Sigma Aldrich).The concentration and purity of the RNA were determined with a NanoDrop™ Lite Spectrophotometer (Thermo Fisher Scientific).Three independent replicates for each experimental condition were prepared.cDNA was synthesized from 1 µg of each RNA sample using PrimeScript RT Reagent Kit with gDNA Eraser (Takara), and the primers used for RT-PCR were designed using Primer3web, version 4.1.0and are listed in Table S2.RT-PCR reactions were performed in 10 µL mixtures containing 50 ng cDNA, 0.3 µM of each primer, and 50% SYBR green (PowerUp SYBR Green Master Mix, Thermo Fisher, Waltham MA), using a Thermo Fisher (QuantStudio3) thermocycler with the following parameters: (i) initial denaturation, 4 min at 95°C; (ii) 40 cycles of .CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprint this version posted January 8, 2024.; https://doi.org/10.1101/2024.01.07.574578 doi: bioRxiv preprint denaturation, primer annealing, and extension (20 s at 95°C, 30 s at 60°C, 30 s at 72 °C ); (iii) production of melting curve, from 50°C to 90°C (rate: 0.58°C every 5 s).The relative amount of mRNA of each gene was determined using the 2 -∆∆Ct formula, where the threshold cycle (Ct) of the target gene in the treated sample is normalized to the reference gene (oprI or rpoD) and the respective value obtained in the untreated bacteria.

Fractionation of P. aeruginosa supernatants
Bacteria were grown in 20 ml of VBMM for 18 hours at 37°C under shaking.Cells were centrifuged at 5000 rpm for 15 min, and supernatants were collected and filtrated using a 0.22 µm syringe filter (Cytiva, Marlborough, MA, USA).The filtrates were acidified with 37% HCl to pH 2.0-3.0,diluted with 0.4 volumes of ethyl acetate, and centrifuged at 5000 rpm for 15 min.Ethyl acetate addition and centrifugation were repeated three times.The organic phases were collected and dried in SC110 Speedvac® Concentrator (Savant Instruments, Holbrook, NY) for up to three hours.The resulting pellets were resuspended 1:20 (vol:vol) methanol:ddH2O for the feeding assays and 1:1 (vol:vol) methanol:ddH2O for spectrophotometric analysis.

zrmA promoter activity assay
The reporter plasmid pzrmApTZ110 (19), carrying the promoter region of zrmA upstream of the lacZ gene, was mobilized from E. coli DH5α into PA14 strains by triparental mating, using E. coli HB101 pRK2013 as the helper strain (21).PA14 exconjugants carrying pzrmApTZ110 were pre-inoculated overnight in LB medium, diluted 1:1000 in E-VBMM, and incubated at 37°C for 18 hours.For each experimental condition, three independent colonies were tested, following the beta-galactosidase activity assay previously described (23).In triplicate, absorbances were measured in Sunrise™ reader (Tecan, Männedorf, Switzerland).

Modeling of PA4064/PA4065 and PA2911-PA2914
The structures of the individual proteins encoded by the P. aeruginosa PA2911-PA2914 gene cluster (UniProt ID: Q9HZT6, Q9HZT5, Q9HZT4, and Q9HZT3) were modeled through the SWISS-MODEL web server (67).The model of the uncharacterized complex PA4064-4065 (UniProt ID: Q9HWW3 and Q9HWW4) was obtained by the Alphafold 2 method (68), applying the AlphaFoldmultimer prediction model, using the full database for the construction of multiple sequence alignments (MSAs) and excluding from the pipeline the 3D structures published before December 31 st , 2000.In this technique, a folded protein is considered a "spatial graph" where residues are the nodes, and edges connect the residues in close proximity.An attention-based neural network system, trained end-to-end, attempts to interpret the structure of this graph while reasoning over the implicit graph has been created in the latest version of AlphaFold used at CASP14 (Critical Assessment of Techniques for Protein Structure Prediction, May-August 2020).

Metal analysis
P. aeruginosa strains were grown in 10 mL of E-VBMM containing trace amounts of transition metals, namely ZnSO4 0.2 μM, FeSO4 0.1 μM, NiSO4 0.1 μM, Co(NO3)2 0.1 μM, CuSO4 0.1 μM, and MnCl2 0.1 μM.Cultures were grown for 18 h at 37°C in 50 mL polypropylene tubes, collected, and pelleted by centrifugation at 6000 rpm for 10 min.The samples were carefully washed with 10 mL of phosphate buffer saline (PBS) containing EDTA 1 mM to remove metals weakly bound to the bacterial surface.Bacterial pellets were dried in SC110 Speedvac® Concentrator and stored at -20°C for further analyses.Metal content in bacterial pellets was determined by an Inductively Coupled Plasma Mass Spectrometer (ICP-MS, model 820-MS; Bruker, Bremen, Germany) equipped with a collision-reaction interface (CRI).Fe and Mn were analyzed using ICP-MS in CRI mode with H2 and He (99.9995% purity; SOL Spa, Monza, Italy) as cell gases; the other elements by ICP-MS in standard mode.The ICP-MS optimized instrumental parameters are summarized in a previous study (69).Multi-element standard solutions (VWR International, Milan, Italy) were used for instrumental calibration curves (seven-point).Yttrium at 0.005 mg L −1 (Panreac Química, Barcelona, Spain) and

Fig 1 .
Fig 1.Schematic representation of the PA2911-PA2914 and PA4063-PA4066 operons.The corresponding gene ID in PA14 strain and a brief description of the function are reported for each ORF.Sources are: a Pseudomonas Genome Database (https://www.pseudomonas.com)and b (30).

Fig 2 .
Fig 2. (A)Transcription of PA4063 and PA2911 in response to Zn availability.RT-PCR on PA2911 and

(
log2Ct) compared to the gene expression in the wild-type strain grown in E-VBMM (control).Statistical analyses were performed by Two-way ANOVA and Bonferroni's multiple comparison test.Asterisks indicate statistically significant differences between all samples and the control (*p<0.05;**p<0.005;****p < 0.0001); hash signs indicate statistically significant differences between the znuAzrmB strain grown in E-VBMM and E-VBMM + Zn ( #### p < 0.0001).(B) Contribution of PA2914 and PA4065 to PA14 growth under conditions of Zn limitation.Growth curves of wild-type, znuAzrmB, and mutant strains carrying the PA2914 deletion or the PA4065 deletion, as indicated in the legends, grown in E-VBMM.Each symbol indicates the mean ± SD of triplicates, and lines represent nonlinear fit according to the Logistic Growth equation.(C) Effect of PA4065 and PA2914 deletions on intracellular Zn request.The beta-galactosidase activity driven by the zrmA promoter was evaluated after 20 hours of growth in E-VBMM in strains carrying plasmid pzrmApTZ110.Each bar represents the mean ± SD of three independent experiments.Statistical significance was calculated by one-way ANOVA and Bonferroni's multiple comparison test.Asterisks indicate statistical significance between wild-type (wt) and mutant strains (* p < 0.05; **** p < 0.0001); hash signs indicate statistical significance between PA4065znuAzrmB and the other mutant strains ( #### p < 0.0001).

2 (
Fig 3) is formed by the assembly of two pairs of subunits.PA4064 indeed codes for the N-terminal nucleotide-binding domains (Fig 3, green and orange), while PA4065 codes for the TM helices and the periplasmic domains (Fig 3, gray and red).The pLDDT values for both domains are notably high (S2 Fig.), indicating the high quality and reliability of the models.These findings suggest that the PA4063-4066 operon does not encode for a metal import system but, more plausibly, for a transporter involved in the export of a molecule contributing to cellular responses to Zn deficiency.

Fig 3 .
Fig 3. Molecular modelling of PA4064-PA4065.Molecular representation of the predicted MacB transporter . Electro Spray Ionization Mass Spectrometry (ESI-MS) analyses of the ethyl acetate fractions from the wild-type, PA4065znuAzrmB and PA2914znuAzrmB strains confirmed the presence of PCH, but failed to detect PCH in the znuAzrmB sample (S4 Fig.).S4 Fig. also reveals that the mass spectrum profile of the supernatant from the znuAzrmB double mutant is extremely simplified compared to the other strains and that in addition to PCH, other metabolites are absent or strongly reduced.To explore whether the difference in PCH secretion between wild-type and znuAzrmB strains might be related to a modulation of gene expression in PCH metabolic pathway, we conducted RT-PCR analyses of the transcription levels of genes involved in PCH regulation (pchR), uptake (fptA and fptX) and synthesis (pchD and pchE) in wild-type and znuAzrmB strains grown in E-VBMM.Results indicated that the transcription of pchD, pchE, fptA, and fptX was significantly downregulated in the znuAzrmB strain compared to the wild-type, while pchR transcription was not significantly affected (Fig 4B).

Fig 5 .
Fig 5. Effects of PCH on intracellular Zn request.(A) The zrmA promoter activity was evaluated in strains

Fig 6 .
Fig 6.Quantification of PCH in cell lysates and cell-free supernatants.Wild-type and mutant strains were

Fig 7 .
Fig 7. Influence of PCH on intracellular Zn request.The zrmA promoter activity was evaluated in znuAzrmB

8 ,
right panel).While the inactivation of znuA does not affect Co content, strains unable to synthesize PCH or pseudopaline displayed a sharp decrease in Co accumulation.This result suggests that PCH and pseudopaline play a role in Co uptake during Zn starvation.Intriguingly, while the Co content of the PA2914znuAzrmB remains comparable to that of all the pchE mutants, the deletion of PA4065 led to a significant and unexpected increase of Co content unless PCH production was abrogated.These findings strongly suggest that PA2914 and PA4065 are involved in the trafficking of the PCH-Co complex in and out of the cell during Zn starvation.

Fig 8 .
Fig 8. Intracellular Zn and Co content.ICP-MS analyses of bacteria grown in E-VBMM with trace metals To investigate the role of PCH in Co accumulation, we conducted growth experiments with the PA4065znuAzrmB and PA4065znuAzrmBpchE mutant strains in E-VBMM, with or without Co supplementation.As shown in Fig 9, Co supplementation caused a noticeable growth delay in the PA4065znuAzrmB mutant, similar to what was observed in the wild-type strain (S5 Fig.), likely explained by the PCHmediated Co accumulation in the cell.In contrast, the quadruple mutant PA4065znuAzrmBpchE,

Fig 9 .
Fig 9. Effect of pchE on Co supplementation of strains carrying the PA4065 deletion.Growth curves of

Fig. 10 .
Fig. 10.Schematic view of P. aeruginosa responses to Zn deficiency.The diagram illustrates the metal

S5Fig.
Effect of Co supplementation on PA14 wild type and znuAzrmB strains.Bacteria were grown in E-VBMM (continuous lines) and E-VBMM + Co(NO3)2 10 M (dotted lines).Each symbol indicates the mean ± SD of triplicates, and lines represent nonlinear fit according to the Logistic Growth equation.S1 Tab.Bacterial strains and plasmids.S2 Tab.List of primers used in this work.S3 Tab.ICP-MS analyses of intracellular Fe content.
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is used for the generation of knock-out mutants are listed in Table