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Article

Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in Pseudomonas putida KT2440

by
Tania Henriquez
1,
Tom Baldow
1,
Yat Kei Lo
1,
Dina Weydert
1,
Andreas Brachmann
2 and
Heinrich Jung
1,*
1
Biozentrum, Mikrobiologie, Ludwig-Maximilians-Universität München, 82152 Martinsried, Germany
2
Biozentrum, Genetik, Ludwig-Maximilians-Universität München, 82152 Martinsried, Germany
*
Author to whom correspondence should be addressed.
Microorganisms 2020, 8(11), 1782; https://doi.org/10.3390/microorganisms8111782
Submission received: 23 October 2020 / Revised: 9 November 2020 / Accepted: 12 November 2020 / Published: 14 November 2020
(This article belongs to the Section Molecular Microbiology and Immunology)
Browse Figures
Versions Notes

Abstract

:
Bacteria must be able to cope with harsh environments to survive. In Gram-negative bacteria like Pseudomonas species, resistance-nodulation-division (RND) transporters contribute to this task by pumping toxic compounds out of cells. Previously, we found that the RND system TtgABC of Pseudomonas putida KT2440 confers resistance to toxic metal chelators of the bipyridyl group. Here, we report that the incubation of a ttgB mutant in medium containing 2,2’-bipyridyl generated revertant strains able to grow in the presence of this compound. This trait was related to alterations in the pp_2827 locus (homolog of mexS in Pseudomonas aeruginosa). The deletion and complementation of pp_2827 confirmed the importance of the locus for the revertant phenotype. Furthermore, alteration in the pp_2827 locus stimulated expression of the mexEF-oprN operon encoding an RND efflux pump. Deletion and complementation of mexF confirmed that the latter system can compensate the growth defect of the ttgB mutant in the presence of 2,2’-bipyridyl. To our knowledge, this is the first report on a role of pp_2827 (mexS) in the regulation of mexEF-oprN in P. putida KT2440. The results expand the information about the significance of MexEF-OprN in the stress response of P. putida KT2440 and the mechanisms for coping with bipyridyl toxicity.

1. Introduction

Pseudomonas species are Gram-negative rod-shaped bacteria that exhibit a highly versatile range of activities, including acting as plant protectants (such as Pseudomonas putida) or animal pathogens (such as Pseudomonas aeruginosa) [1,2]. The bacteria live in changing environments in which they must adapt and respond to stress in order to survive. Resistance-nodulation-division (RND) transport systems contribute to this task by pumping toxic compounds out of cells [3,4]. For example, the expression of mexXY is elevated in the presence of ribosome-targeting antibiotics [5], while the mexCD-oprJ operon is induced by membrane damaging agents [6]. Furthermore, several clinical isolates of P. aeruginosa exhibit the so-called nfxC phenotype that is characterized by expression of the normally quiescent mexEF-oprN operon (conferring resistance to quinolones and chloramphenicol), and the downregulation of oprD (leading to a decreased uptake of imipenem [7]). The phenotype was originally described in P. aeruginosa strain PAO4009 after exposure to norfloxacin [8]). The mutation leading to this phenotype can be located in mexT (coding for a regulator of mexEF-oprN and oprD) [9], mexS (regulator of mexT) [9], mexEF-oprN, oprD, mvaT [10], ampR [11] (the latter two genes code for transcriptional regulators), or in other unknown regions of the genome [12,13]. While most of this information on MexEF-OprN and its regulatory network was derived from clinical isolates of P. aeruginosa, little is known about the role and regulation of the RND system in other species, including P. putida.
In our previous work, we described that in P. putida KT2440 the RND system TtgABC is needed in order to cope with the toxicity of the metal chelating compounds 2,2’-bipyridyl (Bip) and caerulomycin (a 2,2’-bipyridyl-derivaive produced by bacteria and normally found in the environment [14]) [15]. In the absence of a functional transporter, Bip is most likely accumulated inside the cells, where it can sequester copper, iron and other metals, thereby interrupting the normal functioning of metal-dependent enzymes and protein complexes. Indeed, we observed that a ttgB mutant (lacking the inner membrane component of the TtgABC system) had an impaired growth phenotype and showed several metabolic defects in the presence of Bip (such as reduced intracellular ATP levels and inhibition of siderophore production) [15].
In the present work, we report the generation and characterization of second site revertants of a ttgB mutant of P. putida KT2440. Revertants were isolated after prolonged incubation of the ttgB mutant in presence of Bip and were able to grow in the presence of this compound (like P. putida KT2440 wild type strain). The molecular determinants of the phenotype were identified and evaluated by deletion and complementation experiments as well as gene expression analyses. Our results indicate that expression of the genes encoding the RND system MexEF-OprN is responsible for restoring the resistance to Bip in the ttgB mutant revertants. Expression of mexEF-oprN was dependent on alterations in the pp_2827 locus (mexS). The results obtained by these experiments provide insights into the role of MexEF-OprN in stress response and reveal components involved in its regulation in P. putida KT2440.

2. Materials and Methods

2.1. Bacterial Strains and Culture Media

A complete list of strains, plasmids, and oligonucleotides used in this research can be found in Table 1 and Table S1. The strains were cultured in King’s Broth (KB) medium [16] at 30 °C and stored as frozen glycerol stocks. 2,2’-Bipyridyl (Bip) (Sigma) was added to the medium at final concentrations of 0.5 or 1 mM when appropriate. For experiments in P. putida using plasmids, 1 mg/mL ampicillin or 50 μg/mL kanamycin was used. For susceptibility testing, Mueller Hinton (MH) medium was prepared according to the manufacturer’s instructions.

2.2. Colony Morphology Assay

The original protocol from Sakhtah and colleagues [21] was slightly modified. Briefly, overnight cultures were adjusted to an OD600 of 3. Then, 10 μL were spotted onto KB agar plates and KB plus 1 mM Bip and incubated at 30 °C for 24 h. The colonies were photographed under visible and UV light. The final image processing was done with ImageJ [22] and Adobe Illustrator.

2.3. Generation of Mutants and Complemented Strains

All genes were deleted by homologous recombination using the pNPTS138-R6KT suicide vector [23]. Briefly, upstream and downstream regions of the area to be deleted were amplified and fused by PCR. The resulting amplicon was cloned into pNPTS138-R6KT and used to transform the corresponding initial strain (first recombination). This strain was then grown on cetrimide agar plus 10% sucrose in order to select for the second recombination. The mutant strain was screened by PCR and confirmed by DNA sequencing. For complementation, pp_2827 and mexF were amplified by PCR, cloned into the corresponding plasmid (pUCP-Nde and pSEVA224) and used to transform P. putida strains. All oligonucleotides used for amplification are listed in Table S1.

2.4. Growth Curves

Overnight cultures in KB medium were used to inoculate baffled flasks with 35 mL of KB or KB plus 0.5 mM Bip (initial OD600 of ~0.1). The flasks were incubated at 30 °C with continuous shaking at 180 rpm for 8 h. Every 60 min, 1 mL of bacterial culture was taken and used to measure OD600. Each experiment was performed a minimum of three times and the data shown in respective graphs represent the average of all replicates.

2.5. Luciferase Activity Assay

A PmexF::luxCDABE transcriptional reporter gene fusion was generated by PCR amplification of the promoter region of the mexEF-oprN operon and cloning of the resulting fragment into the BamHI and XhoI sites of plasmid pBBR1-MSC5-lux [20]. P. putida strains were transformed with the resulting plasmid pBBR1-MCS5-Pmex::lux. Overnight cultures of the resulting strains were used to inoculate 96-well plate in KB with or without 0.5 mM Bip and 30 μg/mL gentamicin. The final volume of the wells was 100 μL with an initial OD600 of 0.1. The experiment was performed at 30 °C with continuous shaking for 12 h. After 8 h of growth, luminescence was measured and normalized against the OD600 of each strain. Growth and luminescence were measured in a CLARIOstar Plus (BMG LABTECH®, Ortenberg, Germany).

2.6. Susceptibility Testing by Diffusion Method

Bacteria were grown on MH agar plates and then used to prepare an inoculum on saline solution (0.85% w/v NaCl) equivalent to a McFarland 0.5 (OD625 of 0.08–0.13). This bacterial suspension was used to inoculate a new MH plate with a cotton swab. The inoculation was made over the plate in three different directions (to get a complete lawn). Then, susceptibility discs from Thermo Scientific™ Oxoid™ were place over the plate using an antimicrobial susceptibility disc dispenser (Thermo Scientific™ Oxoid™, Waltham, MA, USA) and pressed against the agar with tweezers. For this assay, discs of Gentamicin (10 µg) and chloramphenicol (30 µg) were used. Finally, the plates were incubated for 18 h at 30 °C and the inhibition zone around each antibiotic was measured with a ruler.

2.7. Genomic Sequence Analysis

Genomic DNA of revertant and wild type strains was extracted using the Wizard® SV Genomic DNA Purification System (Promega) and for each sample purity was determined on NanoDrop ND-1000 (PeqLab). Library preparation was performed with 100 ng of genomic DNA each, as quantified on Qubit 2.0 Fluorometer (ThermoFisher Scientific with ds HS Assay Kit), using the Nextera DNA Flex Library Prep Kit (Illumina) according to manufacturer’s instructions. Libraries were quality controlled with DNA High Sensitivity DNA Kit on Bioanalyzer (Agilent) and quantified on Qubit 2.0 Fluorometer (ThermoFisher Scientific with ds HS Assay Kit). Genome sequencing was performed in the Genomics Service Unit (LMU Biocenter, Munich, Germany) on Illumina MiSeq with v3 chemistry (2× 250 bp paired-end sequencing). Genome assemblies and variant detection were performed on CLC Genomics Workbench 9 (Qiagen). The data have been deposited with links to BioProject accession number PRJNA670853 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/).

2.8. Statistical Analysis

GraphPad/Prism 8 was used for statistical analysis. One-way ANOVA with Dunnett’s multiple comparison and t-test were performed as appropriated. All experiments were performed a minimum of three times.

3. Results

3.1. Bip-Driven Generation of Second Site Revertant Strains from the ttgB Mutant

In our previous research, we have described that deletion of ttgB creates a growth defect in presence of Bip and inhibits the production of the siderophore pyoverdine [15]. This phenotype was most likely related to the accumulation of Bip inside the cell in absence of a functional TtgABC system. In the course of subsequent experiments, we observed that after prolonged incubation in presence of 1 mM Bip, the ΔttgB strain derived from P. putida KT2440 started to grow. More specifically, in a colony morphology assay on KB agar plates supplemented with 1 mM Bip, P. putida KT2440 (wild type) formed a large colony, while the ΔttgB mutant yielded very small colonies in the inoculated area within 24 h (Figure 1A). Furthermore, when exposed to UV light, wild type colonies and the small colonies derived from the ΔttgB mutant were fluorescent, suggesting that the cells produced the siderophore pyoverdine under the indicated experimental conditions (Figure 1A). We wondered if the small colonies were the result of very slow growth of the mutant or due to compensating mutations elsewhere in the genome. In order to answer this question, bacteria of the small colonies were isolated on KB agar plates without Bip and the colony morphology assay was repeated. Contrary to the original ΔttgB mutant, all isolates formed large colonies in the presence of Bip, similar to wild type. More quantitative analyses of the growth dynamics in liquid KB medium containing 1 mM Bip revealed that the growth of the isolated strains was faster compared to the ΔttgB mutant and even similar to the wild type (Figure 1B). In the absence of Bip, wild type, original ΔttgB mutant and isolated strains grew equally well in KB medium (Figure 1B). Altogether, these results suggest that (an) additional mutation(s) compensate(s) for the deletion of ttgB in the isolated strains (called hereafter Revertants A, B and C) and restored the resistance against Bip.

3.2. Alteration in pp_2827 Rescues Growth of the ttgB Mutant in Presence of Bip

To obtain information on the molecular basis of the second site mutation in Revertants A, B and C, a genomic sequence analysis was performed. In Revertant A, a single base substitution was found in locus pp_2827 (A841G), predicted to encode a 340 amino acid oxidoreductase/dehydrogenase [24,25]. The alteration would lead to the substitution of the amino acid isoleucine by valine (I281V) in the putative zinc-binding cassette of the predicted enzyme (Supplementary Material, Figure S1A). In Revertant B, we found an insertion of 17-kb-long transposon Tn4652 (Tsuda & Iino, 1987) after position 891 in pp_2827, leading to a replacement of the 43 C-terminal amino acids. This fusion also disrupts the putative zinc-binding cassette (Supplementary materials, Figure S1A). In Revertant C, a 250 kb genomic region between pp_3382 and pp_3585 is present in multiple copies (Supplementary Material, Figure S1B). This region is flanked by pp_3381 and pp_3586, two copies of the IS110-like element ISPpu9, which is present 7 times in the P. putida KT2440 genome [26]. We focused on Revertant A in subsequent analyses.
To further investigate the role of pp_2827 in the revertant phenotype, we deleted pp_2827 in the ΔttgB mutant. Our results indicated that the growth advantage of the ΔttgB Δpp_2827 strain in presence of Bip was similar to the Revertant A strain (Figure 2A). In parallel, deletion of pp_2827 in the wild type strain did not result in any growth difference (Figure 2A). Obviously, the functional TtgABC system of the wild type was able to cope with Bip toxicity. In accordance with these results, the complementation of pp_2827 in the ΔttgB Δpp_2827 strain made the bacterium susceptible to the toxicity of Bip (Figure 2B). Taken together, these results indicate that alterations in the locus pp_2827 can rescue growth of the ttgB mutant in presence of Bip.

3.3. Alterations in pp_2827 Confer Bip Resistance by Stimulating Expression of mexEF-oprN

In order to further explore the resistance mechanism to Bip in Revertant A, we analyzed possible links of pp_2827 to other genes. In P. aeruginosa, the locus orthologous to pp_2827 is pa2491 (mexS), which together with the regulator gene pa2492 (mexT) is located immediately upstream of the mexEF-oprN operon (pa2493–pa2495) [27]. The gene products of both mexS and mexT were previously implicated in the regulation of the expression of mexEF-oprN [9,28,29], an RND transporter that is normally quiescent in P. aeruginosa [30]. In P. putida KT2440, pp_2827 and pp_2826 (mexT), and the loci orthologous to mexEF-oprN (pp_3425–pp_3427) are located far away from each other in the genome [24,25]. The role of pp_2827 in P. putida, to our knowledge, has not been explored yet.
Thus, we wondered if the observed phenotype in the revertant strains was due to pp_2827 mutation influencing the activity of the MexEF-OprN efflux system. To that end, we analyzed the expression of mexEF-oprN in these strains through a transcriptional fusion that contained the promoter of mexE fused to the luxCDABE operon (PmexE::luxCDABE) in plasmid pBBR1-MSC5-lux. Our results showed that after 8 h of growth, the relative luminescence (normalized against OD600) of the Revertant A strain was much higher than the one from the wild type strain (Figure 3), indicating that the mexEF-oprN promoter was active.
Additionally, the activity of the MexEF-OprN system was indirectly observed through an increased resistance of Revertant A strain to chloramphenicol, while there was no effect in the susceptibility to gentamicin (Figure 4). These results fit to the previously observed MexEF-OprN-mediated resistance pattern of P. aeruginosa [31].
In P. aeruginosa, the impairment of mexS, and the concomitant activation of mexT, increases the expression not only of mexEF-oprN but also affects other genes (e.g., it represses oprD) [32]. Therefore, we wondered whether the Revertant A phenotype in P. putida was indeed due to the enhanced expression of the RND system or due to another element regulated by this network. Thus, we deleted the inner membrane component of the RND system, mexF, in the ΔttgB Δpp_2827 strain and in Revertant A. Our results showed that both strains lost the resistance to Bip when mexF was deleted (Figure 5). On the contrary, in the absence of Bip all strains grew equally well (Figure 5). This indicates that a functional MexEF-OprN system is required for the phenotype of Revertant A. These results were corroborated by complementation experiments, where the expression of mexF from a plasmid was able to rescue the growth of the strains similarly as the plasmid-based expression of ttgB (Supplementary Materials, Figure S2). Altogether the results indicate that in Revertant A enhanced expression of mexEF-oprN is necessary to cope with Bip toxicity in the absence of a functional TtgABC.

3.4. The mexEF-oprN Operon is also Upregulated in Revertants B and C

To obtain more information on the molecular basis of Bip resistance in the other revertant strains, we analyzed expression of the mexEF-oprN operon also in Revertant B and C and the engineered ΔttgB Δpp_2827 strain using plasmid pBBR1-MCS5-Pmex::lux. Bioluminescence measurements revealed that all three strains expressed mexEF-oprN, although to different degrees (Figure 6). The higher expression of mexEF-oprN in the Revertant B can also be seen indirectly through the increase in the resistance to chloramphenicol (Figure S3). For Revertant B, upregulation of mexEF-oprN was expected since in the strain pp_2827 was altered (transposon insertion) similar to what was observed for Revertant A (point mutation in pp_2827). However, the level of upregulation of the operon was about 20fold higher than in Revertant A and at about the same level as for the engineered ΔttgB Δpp_2827 strain (Figure 6). The mechanism for the moderate upregulation of the mexEF-oprN operon in Revertant C remained enigmatic.

4. Discussion

While inactivation of the RND system TtgABC renders P. putida KT2440 sensitive to metal ion chelators of the bipyridyl group [15], we describe here that incubation of a ttgB mutant of P. putida KT2440 in presence of Bip rapidly leads to the generation of second site revertant strains. Since intracellularly accumulated Bip interferes with the cellular homeostasis for iron, cupper and potentially other metal ions required for central metabolic pathways and the respiratory chain, a rapid cellular response compensating the loss of TtgABC activity is not surprising. Known responses to intracellular metal ion limitation in bacteria include the stimulation of the uptake of metal ions, mobilization of limited metal ions from intracellular storage pools, and substitution of metal-dependent enzymes and pathways that function independently of the respective metal ion [33]. None of these possibilities seem to be used by P. putida KT2440 under our test conditions. Instead, the primary cause of intracellular ion limitation, the chelator Bip, seems to be removed from cells via an alternative RND system, MexEF-OprN. The latter system is known to be quiescent in P. aeruginosa [30], and we showed that the respective operon is also not expressed in P. putida KT2440, independent of the presence of Bip in the culture medium. However, in all three revertants generated from the ttgB mutant and analyzed here, mexEF-oprN was expressed constitutively. Deletion of mexF and plasmid-based complementation confirmed that the RND system is indeed required and most likely sufficient for rescuing growth of the ΔttgB strain in the presence of Bip.
Identification of a point mutation and transposon insertion in the locus pp_2827 in the isolated Revertant A and B strains, respectively, and subsequent deletion and complementation analyses with pp_2827 demonstrate that PP_2827 represses the transcription of the mexEF-oprN operon. The gene product has been annotated as a zinc-dependent oxidoreductase/alcohol dehydrogenase in the genome of P. putida [24,25]. In a previous report, pp_2827 and mexEF-oprN were identified as belonging to the PhhR regulon that is made responsible for the activation of genes essential for phenylalanine degradation, phenylalanine homeostasis and other genes of unknown function [34]. However, to the best of our knowledge, this is the first report of an involvement of pp_2827 in the regulation of mexEF-oprN expression in P. putida. The locus adjacent to pp_2827, pp_2826, encodes a LysR-type transcriptional regulator termed MexT that was previously shown to stimulate mexEF-oprN expression in P. putida KT2440 [34]. Taken together, the results suggest a regulatory cascade, in which pp_2827 (MexS) inhibits the expression of the mexEF-oprN operon by repressing mexT. The mechanism of the repression is not known. The putative oxidoreductase MexS is not predicted to contain a DNA binding domain. So far, it can only be speculated that the enzyme produces a metabolite, alters the redox state of another compound or protein, or physically interacts with other proteins leading to repression of mexT. In this context, the relatively low levels of mexEF-oprN expression observed for Revertant A relative to Revertant B and the engineered ΔttgBΔpp_2827 strain may be due to an only partial inactivation of the putative oxidoreductase (MexS) in Revertant A, while in the latter two strains the enzyme is completely inactive. In fact, the point mutation in Revertant A causes an amino acid substitution (I281V) in the predicted zinc binding domain of the enzyme that may alter enzyme kinetics without abolishing activity. This is the first report showing that an alteration at this site can stimulate mexEF-oprN expression. In clinical isolates and laboratory generated spontaneous mutants of P. aeruginosa with elevated expression of mexEF-oprN, positions such as the N-terminal or the zinc-binding domain were altered in MexS [29,35]. While MexS-dependent regulation of mexEF-oprN is dependent of MexT in P. aeriginaosa [29], another publication reports that MexS can in principal also have inhibitory effects on gene expression independently of MexT [36].
The molecular mechanisms behind the upregulation of mexEF-oprN in Revertant C may be due to transposon-based gene duplication events. Our genome comparisons do not hint at changes in the regions of mexS, mexT, mexEF-oprN, mvaT, ampR, which have been reported to be causative for the nfxC phenotype of P. aeruginosa [9,10,11]. This result is in accordance with previous reports in clinical strains of P. aeruginosa, where significant percentages of the screened strains had a nfxC phenotype with unknown mechanisms for the induction of mexEF-oprN expression [12]. Furthermore, the relatively low level of expression of mexEF-oprN in Revertant C may be explained by a functional pp_2827 (MexS) that most likely inhibits upregulation of the operon. Furthermore, for Revertant C it is not clear whether this upregulation is indeed responsible for Bip resistance. Although the large amplified genomic region (pp_3382 and pp_3585) contains also the mexEF-oprN operon, an involvement of other molecular players cannot be excluded.
All three revertant strains analyzed here exhibit a behavior similar to the nfxC phenotype of P. aeruginosa [12,37], including the expression of mexEF-oprN and a higher resistance to chloramphenicol. In the native environment of the soil bacterium P. putida KT2440, MexEF-OprN could be involved in the detoxification of natural bipyridyls such as caerulomycin and collismycin that are produced by other bacteria [14,38].
It is interesting to note how quickly (in less than 24 h of exposure) incubation in presence of Bip transformed the ttgB mutant into a much more resistant strain (increased resistance to Bip, chloramphenicol and probably other bipyridyls) by second site mutation. Two of the three revertants most likely stem from transposon activity, which at least for Tn4652 is known to be induced in P. putida under stress conditions [39]. From a clinical point of view, this reinforces the necessity to discover effective inhibitors of RND efflux pumps to avoid the fast appearance of spontaneous resistance. Altogether, these results increase the information about the role of MexEF-OprN in stress response in P. putida KT2440 and extend the role of MexS in Pseudomonas species.

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-2607/8/11/1782/s1, Figure S1: Mutations in Revertant A, B and C strains, Figure S2: Phenotype complementation with ttgB and mexF in the Revertant A ΔmexF and ΔttgB Δpp_2827 ΔmexF strains, Figure S3: Susceptibility of Revertant B to chloramphenicol, Table S1: List of primers used in this study.

Author Contributions

Conceptualization, T.H. and H.J.; methodology, T.H. and H.J.; software, A.B., T.H., T.B., Y.K.L., D.W. and H.J.; validation, H.J. and T.H.; formal analysis, A.B., T.H., T.B., Y.K.L., D.W. and H.J.; investigation, T.H., T.B., Y.K.L., and D.W.; resources, H.J., A.B. and T.H.; data curation, H.J., A.B. and T.H.; writing—original draft preparation, H.J. and T.H.; writing—review and editing, H.J., A.B. and T.H.; visualization, H.J., A.B. and T.H.; supervision, H.J. and T.H.; project administration, H.J.; funding acquisition, H.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research and the APC were funded by the Deutsche Forschungsgemeinschaft through grants JU333/5-1, 2 (SPP1617) and JU333/6-1.

Acknowledgments

We thank Michelle Eder for excellent technical assistance.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Second site mutation rescues growth of ΔttgB strain in presence of Bip. (A). Overnight cultures were spotted onto King’s Broth (KB) agar plates with or without supplementation with 1 mM Bip and incubated at 30 °C. After 24 h, pictures were taken using visible and UV light. The red arrows indicate the position of some of the revertant colonies that are better visible in UV light. (B) Growth curves of wild type, ΔttgB and Revertant A strain (red) were performed in KB and KB plus 0.5 mM Bip for 8 h at 30 °C and continuous shaking (180 rpm). One ml of culture was taken and used to record its OD600 every 60 min. Experiments were performed a minimum of three times.
Figure 1. Second site mutation rescues growth of ΔttgB strain in presence of Bip. (A). Overnight cultures were spotted onto King’s Broth (KB) agar plates with or without supplementation with 1 mM Bip and incubated at 30 °C. After 24 h, pictures were taken using visible and UV light. The red arrows indicate the position of some of the revertant colonies that are better visible in UV light. (B) Growth curves of wild type, ΔttgB and Revertant A strain (red) were performed in KB and KB plus 0.5 mM Bip for 8 h at 30 °C and continuous shaking (180 rpm). One ml of culture was taken and used to record its OD600 every 60 min. Experiments were performed a minimum of three times.
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Figure 2. Alteration in the locus pp_2827 affects growth of the ΔttgB mutant in presence of Bip. (A) Growth curve of wild type, ΔttgB, Δpp_2827 and strains with revertant phenotype (in red: Revertant A and the engineered ΔttgB Δpp_2827 strain). (B) Expression of pp_2827 from plasmid pUCP-pp_2827 reduces growth of the ΔttgB Δpp_2827 strain. For (A,B), growth curves were recorded in KB and KB plus 0.5 mM Bip at 30 °C and continuous shaking (180 rpm) for 8 h. Every 60 min, 1 mL of culture was taken and used to measure the OD600. Experiments were performed a minimum of three times.
Figure 2. Alteration in the locus pp_2827 affects growth of the ΔttgB mutant in presence of Bip. (A) Growth curve of wild type, ΔttgB, Δpp_2827 and strains with revertant phenotype (in red: Revertant A and the engineered ΔttgB Δpp_2827 strain). (B) Expression of pp_2827 from plasmid pUCP-pp_2827 reduces growth of the ΔttgB Δpp_2827 strain. For (A,B), growth curves were recorded in KB and KB plus 0.5 mM Bip at 30 °C and continuous shaking (180 rpm) for 8 h. Every 60 min, 1 mL of culture was taken and used to measure the OD600. Experiments were performed a minimum of three times.
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Figure 3. Promoter activity of the mexEF-oprN operon in P. putida KT2440 in presence of Bip. The transcriptional gene fusion using the lux reporter was generated by cloning the promoter region of mexEF-oprN into the BBR1-MCS5-lux plasmid. For the luciferase assay, overnight cultures of the transformed strains were grown in KB in presence of 0.5 mM Bip and 30 μg/mL gentamicin. After 8 h of growth, the relative light units (RLU) were normalized against the OD600 of the cultures. Experiments were performed a minimum of three times.
Figure 3. Promoter activity of the mexEF-oprN operon in P. putida KT2440 in presence of Bip. The transcriptional gene fusion using the lux reporter was generated by cloning the promoter region of mexEF-oprN into the BBR1-MCS5-lux plasmid. For the luciferase assay, overnight cultures of the transformed strains were grown in KB in presence of 0.5 mM Bip and 30 μg/mL gentamicin. After 8 h of growth, the relative light units (RLU) were normalized against the OD600 of the cultures. Experiments were performed a minimum of three times.
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Figure 4. Susceptibility of the Revertant A strain to chloramphenicol. Susceptibility of wild type ΔttgB and Revertant A strains was tested against chloramphenicol in Mueller Hinton (MH) medium through disc diffusion method. Plates were incubated at 30 °C for 18 h, and the halo diameter was measured. Experiments were performed a minimum of three times.
Figure 4. Susceptibility of the Revertant A strain to chloramphenicol. Susceptibility of wild type ΔttgB and Revertant A strains was tested against chloramphenicol in Mueller Hinton (MH) medium through disc diffusion method. Plates were incubated at 30 °C for 18 h, and the halo diameter was measured. Experiments were performed a minimum of three times.
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Figure 5. Growth of ΔttgB-derivative strains in presence of Bip is due to MexEF-OprN activity. Bacterial growth was analyzed through growth curves in KB medium supplemented with 0.5 mM Bip at 30 °C with continuous shaking (180 rpm). To that end, overnight cultures were used to inoculate 35 mL of medium (initial OD600 of 0.1). Every 60 min, OD600 was measured. Strains with phenotype similar to the original ΔttgB strain (ΔttgB Δpp_2827 ΔmexF and Revertant A ΔmexF) are shown in red. Data are presented as an average of three independent experiments.
Figure 5. Growth of ΔttgB-derivative strains in presence of Bip is due to MexEF-OprN activity. Bacterial growth was analyzed through growth curves in KB medium supplemented with 0.5 mM Bip at 30 °C with continuous shaking (180 rpm). To that end, overnight cultures were used to inoculate 35 mL of medium (initial OD600 of 0.1). Every 60 min, OD600 was measured. Strains with phenotype similar to the original ΔttgB strain (ΔttgB Δpp_2827 ΔmexF and Revertant A ΔmexF) are shown in red. Data are presented as an average of three independent experiments.
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Figure 6. Promoter activity of the mexEF-oprN operon in P. putida KT2440 in presence of Bip. Overnight cultures of the strains containing the transcriptional fusion of the promoter of mexEF-oprN and the luxCDABE operon (in plasmid pBBR1-MCS5-lux) were grown in KB in presence of 0.5 mM Bip and 30 μg/mL gentamicin. After 8 h, relative light units (RLU) were normalized against the OD600. Data are presented as an average of at least three biological replicates.
Figure 6. Promoter activity of the mexEF-oprN operon in P. putida KT2440 in presence of Bip. Overnight cultures of the strains containing the transcriptional fusion of the promoter of mexEF-oprN and the luxCDABE operon (in plasmid pBBR1-MCS5-lux) were grown in KB in presence of 0.5 mM Bip and 30 μg/mL gentamicin. After 8 h, relative light units (RLU) were normalized against the OD600. Data are presented as an average of at least three biological replicates.
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Table
Table 1. List of strains and plasmids used in this study.
Table 1. List of strains and plasmids used in this study.
Name (Strains)DescriptionSource
Wild type (WT) Pseudomonas putida KT2440[17]
ΔttgBDerived from strain KT2440 by deletion of pp_1385[15]
Revertant A, B and CSpontaneous second site revertants of ΔttgB strainThis work
ΔttgB Δpp_2827Derived from the ΔttgB strain by deletion of pp_2827This work
Δpp_2827Derived from strain KT2440 by deletion of pp_2827This work
Revertant A ΔmexFDerived from Revertant A strain by deletion of pp_3426This work
ΔttgB Δpp_2827 ΔmexFDerived from the ΔttgB Δpp_2827 strain by deletion of pp_3426This work
Name (plasmid)DescriptionSource
pUCP (pUCP-Nde)pUCP-NdeI (AmpR) shuttle vector[18]
pUCP-ttgBDerived from pUCP by cloning the ttgB gene from wild type strain into the multicloning site[15]
pUCP-pp_2827Derived from pUCP by cloning pp_2827 from wild type strain into the multicloning siteThis work
pSEVA224KmR; pSEVA221 derivative with lacIq/Ptrc expression system[19]
pSEVA224-mexFpSEVA224 derivative with pp_3426 cloned into the multicloning siteThis work
pBBR1-MCS5-luxpBBR1-based plasmid containing promoter-less luxCDABE, and the aacC1 gene (GenR)[20]
pBBR1-MCS5-Pmex::luxpBBR1-MCS5-lux derivative containing PmexE::luxCDABE This work
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Henriquez, T.; Baldow, T.; Lo, Y.K.; Weydert, D.; Brachmann, A.; Jung, H. Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in Pseudomonas putida KT2440. Microorganisms 2020, 8, 1782. https://doi.org/10.3390/microorganisms8111782

AMA Style

Henriquez T, Baldow T, Lo YK, Weydert D, Brachmann A, Jung H. Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in Pseudomonas putida KT2440. Microorganisms. 2020; 8(11):1782. https://doi.org/10.3390/microorganisms8111782

Chicago/Turabian Style

Henriquez, Tania, Tom Baldow, Yat Kei Lo, Dina Weydert, Andreas Brachmann, and Heinrich Jung. 2020. "Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in Pseudomonas putida KT2440" Microorganisms 8, no. 11: 1782. https://doi.org/10.3390/microorganisms8111782

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