# Developing Biosensors for Specific Assessment of Trans-Translation in Pseudomonas aeruginosa

Bastien L'hermitte, Thomas Chauvet, Sylvie Georgeault-Daguenet, Nicolas Le Yondre, Philippe Jehan, Reynald Gillet, Christine Baysse

► To cite this version:

Bastien L'hermitte, Thomas Chauvet, Sylvie Georgeault-Daguenet, Nicolas Le Yondre, Philippe Jehan, et al.. Developing Biosensors for Specific Assessment of Trans-Translation in Pseudomonas aeruginosa. ACS Synthetic Biology, 2025, 10.1021/acssynbio.4c00801. hal-04995277

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https://univ-rennes.hal.science/hal-04995277v1

Submitted on 18 Mar 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Developing Biosensors for Specific Assessment of

*Trans*-translation in *Pseudomonas aeruginosa*

Bastien L'Hermitte¹, Thomas Chauvet¹, Sylvie Georgeault-Daguenet¹, Nicolas Le Yondre², Philippe

Jehan², Reynald Gillet¹* and Christine Baysse¹*

¹ University of Rennes, CNRS, Institut de Génétique et Développement de Rennes (IGDR) UMR6290,
35000 Rennes, France

² University of Rennes, CNRS, Centre Régional de Mesures Physiques de l'Ouest (CRMPO) ScanMAT
UAR 2025, 35000 Rennes, France

*Co-corresponding authors: reynald.gillet@univ-rennes.fr ; christine.baysse@univ-rennes.fr

Abstract

We developed two *Pseudomonas aeruginosa* biosensors to detect *trans*-translation inhibitors in this medically relevant pathogen. These biosensors leverage the red fluorescence produced by the accumulation of protoporphyrin IX, the substrate of ferrochelatase. The first biosensor monitors tmRNA-SmpB-mediated tagging and degradation of ferrochelatase, while the second serves as a control by
tracking ClpP1-mediated proteolysis and porphyrin biosynthesis. Both biosensors were tested in wild-
type and mutant backgrounds, and red fluorescence was measured relative to absorbance at 600 nm in
microtiter plates. The results confirmed a link between fluorescence and *trans*-translation or proteolysis
activity. These biosensors offer a promising tool for high-throughput screening of *trans*-translation
inhibitors in *P. aeruginosa*.

**Keywords:** *Trans*-translation, *Pseudomonas aeruginosa*, Biosensors, Antimicrobials, Porphyrin, Ferrochelatase

# 1 Introduction

The World Health Organization (WHO) has designated seven “ESKAPEE” pathogenic bacteria (*Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Enterobacter spp.*, and *Escherichia coli*) as critical targets for drug development. Indeed, these seven highly virulent and antibiotic-resistant bacteria are the leading cause of hospital-acquired infections worldwide. The resistance of *P. aeruginosa* to aminoglycosides, quinolones, and β-lactams is of particular concern¹. Moreover, due to the emergence of carbapenem-resistant strains, the WHO has further identified carbapenem-resistant *P. aeruginosa* as one of three bacterial species for which there is a critical need for new antibiotics²,³.

*Trans*-translation is the one of molecular mechanisms involved in the release of ribosomes which find
themselves blocked on mRNAs without stop codons. The process facilitates the degradation of these
faulty mRNAs and proteins and promotes a canonical recycling of ribosomes⁴. This major rescue
pathway has been identified in more than 99 % of bacterial genomes, and it is mediated by transfer-
messenger RNA (tmRNA) associated with the SmpB protein⁵. During the process, incomplete nascent
peptides are tagged for proteolysis thanks to the messenger-like domain (MLD) of the tmRNA.

Problematic mRNAs are then degraded, and the previously stalled ribosomes are recycled⁶. In many
bacterial species, this control machinery is essential for survival, thus it is an excellent target for
antimicrobial compounds⁷,⁸. For species in which this system is not essential, the deletion generally
diminished the virulence, antibiotic tolerance, and, more globally, stress resistance⁹,¹⁰. It may even
prevent the resuscitation of persister cells, as has been demonstrated for *Escherichia coli* ¹¹.

*P. aeruginosa* possesses the canonical tmRNA/SmpB *trans*-translation system, but it also has genes encoding for putative alternative ribosome rescue factors ArfA and ArfB. Therefore, an absence of *trans*-translation does not affect its viability under physiological conditions, but rather alters its virulence and ability to adapt to stressful conditions. Indeed, it has been reported that the *trans*-translation system is crucial for its tolerance to azithromycin and several aminoglycoside antibiotics⁷. In addition, the expression of the genes encoding tmRNA (*ssrA*) and SmpB increased following exposure to tobramycin, suggesting a potential role of *trans*-translation in the pathogen’s adaptation to antibiotics¹².

While high-throughput *in vitro* screening assays to identify *trans*-translation inhibitory molecules in
bacteria of the ESKAPEE group have been recently developed, *in vivo* screening systems in living
bacterial cells has only been developed for *E. coli*¹³⁻¹⁶. These biosensors are based on replicative plasmids
in *E. coli* which encode luciferase, mCherry, or GFP reporters. These systems require ampicillin for
plasmid maintenance, and it can only be used in bacteria that do not naturally produce green or red
fluorescence. Therefore, these systems cannot be adapted to *P. aeruginosa* because it is inherently
resistant to many antibiotics and it can naturally produce green fluorescence. This fluorescence is
particularly strong in iron-deficient environments due to the synthesis of the siderophores pyoverdine
and pyochelin¹⁷. *P. aeruginosa* can also produce red fluorescence when porphyrin derivatives
accumulate. This is detectable in a wild-type strain on medium supplemented with high concentrations of γ-aminolevulinic acid, a precursor in the heme biosynthetic pathway¹⁸. Protoporphyrin IX, one of the red-fluorescent porphyrins, is the direct precursor of a non-fluorescent molecule, heme. Its transformation into heme relies on the activity of ferrochelatase, which is encoded by the *hemH* gene (also known as *ppfC*). Since a *hemH* mutant of *Pseudomonas fluorescens*, a phylogenetically related species, produces a red fluorescence that is easily detectable in colonies and measurable in lysate ¹⁹ (excitation of 405 nm, emission at 630 nm), this led us to target this gene in *P. aeruginosa*.

We therefore decided to exploit the ability of *P. aeruginosa* to produce an endogenous red fluorescent pigment to construct *P. aeruginosa* biosensor strains that can be used to monitor *trans*-translation, without the use of a plasmid and a maintenance pressure based on antibiotic resistance.

# 2 Material and Methods

## 2.1 Strains, plasmids, primers, and growth conditions

All the strains, plasmids and primers are listed in table 1. *E. coli* strains used for plasmid construction have been cultivated in LB broth (LB Lennox: peptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, pH 7) and LB agar medium containing either gentamicin (10 µg/mL), ampicillin (100 µg/mL) or chloramphenicol (20 µg/mL). *P. aeruginosa* strains have been grown in LB, casamino acids medium (CAA: casein peptone 5 g/L, K₂HPO₄ 0.9 g/L, MgSO₄x7H₂O 0.25 g/L), CAA agar, supplemented with sucrose (15 % w/v) if necessary and CN agar for *Pseudomonas* (Grosseron). Gentamicin (100 µg/mL), carbenicillin (300 µg/mL) or chloramphenicol (100 µg/mL) were added if needed. For solid media, 1.5 % (w/v) agar was used.
Biosensor testing medium 1 (Btm1) is composed of CAA supplemented with 40 µM of FeSO₄, 0.5 mM aminolevulinic acid, 10 µM isopropyl β-D-1-thiogalactopyranoside (IPTG). Biosensor testing medium 2 (Btm2) is identical to Btm1 but with the addition of 6.1 µM hemin. For complementation assays, 10 mM L-arabinose was added to either medium.

<table><caption>**Table 1:** strains, plasmids and primers</caption><thead><tr><th>Strain</th><th>Description</th><th>Origin</th></tr></thead><tbody><tr><td><i>Escherichia coli</i></td><td></td><td></td></tr><tr><td>DH5α</td><td><i>fhuA2 lacΔU169 phoA glnV44 Φ80' lacZΔM15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i></td><td>New England Biolabs</td></tr><tr><td>SM10</td><td><i>leu RP4-2 TC::Mu Kan<sup>R</sup></i></td><td><sup>20</sup></td></tr><tr><td><i>Pseudomonas aeruginosa</i></td><td></td><td></td></tr><tr><td>PA14<br>(DSM19882)</td><td>Wild type strain of <i>P. aeruginosa</i></td><td>DSMZ</td></tr><tr><td>Δ<i>hemH</i></td><td>PA14 deletion of <i>hemH</i> (PA14_61580)</td><td>This study</td></tr><tr><td>Δ<i>ssrA</i></td><td>PA14 deletion of <i>ssrA</i> (4749731-4750083)</td><td>This study</td></tr><tr><td>Δ<i>smpB</i></td><td>PA14 deletion of <i>smpB</i> (PA14_63060)</td><td>This study</td></tr><tr><td>Δ<i>clpP1</i></td><td>PA14 deletion of <i>clpP1</i> (PA14_41240)</td><td>This study</td></tr><tr><td>Bios1</td><td>PA14 substitution of <i>hemH</i> with <i>hemH-NS</i> downstream <i>lacIq-Ptac-lacO</i> fragment from pMMB67EH, containing an IPTG inducible promoter</td><td>This study</td></tr><tr><td>Bios2</td><td>PA14 substitution of <i>hemH</i> with <i>hemH-tmRNA</i> tag downstream <i>lacIq-Ptac-lacO</i> fragment from pMMB67EH, containing an IPTG inducible promoter</td><td>This study</td></tr><tr><th>Plasmid</th><th>Description</th><th>Origin</th></tr></tbody></table>
<table><tr><td>pEXG2</td><td>Integrating suicide plasmid for P. aeruginosa, Gent<sup>R</sup>, ColE1 Ori, with sacB for sucrose counterselection</td><td>21</td></tr><tr><td>pMMB67EH</td><td>Broad range expression vector containing an IPTG inducible promoter (lacIq-Ptac-lacO) for P. aeruginosa, Amp<sup>R</sup>, RSF1010 Ori</td><td>22</td></tr><tr><td>pGLO</td><td>GFP encoding plasmid under the control of the pBAD-ARA L-arabinose inducible promoter, pMB1Ori, Amp<sup>R</sup></td><td>Biorad</td></tr><tr><td>pEX-K168</td><td>Cloning vector, Kan<sup>R</sup>, pMB1 Ori</td><td>Eurofins</td></tr><tr><td>pBR325</td><td>Cloning vector, Cm<sup>R</sup>, Amp<sup>R</sup>, Tet<sup>R</sup>, ColE1 Ori</td><td>23</td></tr><tr><td>pKO-ssrA</td><td>pEXG2 containing flanking sequences of ssrA</td><td>This study</td></tr><tr><td>pKO-smpB</td><td>pEXG2 containing flanking sequences of smpB</td><td>This study</td></tr><tr><td>pKO-clpP1</td><td>pEXG2 containing flanking sequences of clpP1</td><td>This study</td></tr><tr><td>pKO-hemH</td><td>pEXG2 containing flanking sequences of hemH with pBR325 cat gene</td><td>This study</td></tr><tr><td>pcomp-ssrA</td><td>pMMB67EH based complementation plasmid containing ssrA gene, downstream pBAD-ARA fragment, containing an L-arabinose inducible promoter from pGLO plasmid</td><td>This study</td></tr><tr><td>pcomp-smpB</td><td>pMMB67EH based complementation plasmid containing smpB gene, downstream pBAD-ARA fragment, containing an L-arabinose inducible promoter from pGLO plasmid</td><td>This study</td></tr><tr><td>pcomp-clpP1</td><td>pMMB67EH based complementation plasmid containing clpP1 gene, downstream pBAD-ARA fragment, containing an L-arabinose inducible promoter from pGLO plasmid</td><td>This study</td></tr><tr><td>psynth_Bios1</td><td>Synthetic construct containing a part of the Bios2 from the RBS to the downstream homology sequence of hemH using pEX-K168 as backbone</td><td>This study</td></tr><tr><td>psynth_Bios2</td><td>Synthetic construct containing hemH with stop codon substituted with tmRNA-tag coding sequence using pEX-K168 as backbone</td><td>This study</td></tr><tr><td>pBios1-INT</td><td>pEXG2 containing the parts of the Bios1 construction</td><td>This study</td></tr><tr><td>pBios1</td><td>pEXG2 containing the Bios1 construction</td><td>This study</td></tr></table>
<table><tr><td>pBios2</td><td>pEXG2 containing the Bios2 construction</td><td>This study</td></tr><tr><td>pantisens_ssrA_1</td><td>pMMB67EH based plasmid containing the gene encoding an antisense RNA targeting the MLD part of the tmRNA, the under the dependence of pBAD-ARA inducible promotor of pGLO</td><td>This study</td></tr><tr><td>pantisens_ssrA_2</td><td>pMMB67EH based plasmid containing the gene encoding an antisense RNA targeting the MLD part of the tmRNA in transcriptional fusion with the MicC expression platform, the under the dependence of pBAD-ARA inducible promotor of pGLO</td><td>This study</td></tr><tr><td>Primer</td><td>Sequence</td><td>Target</td></tr><tr><td>PA14_H1-hemH-H2_Hind3_1</td><td>CCCAAGCTTCCGTTCATAAGGCACCA</td><td>pGEM-T Easy-based intermediary construct</td></tr><tr><td>PA14_H1-hemH-H2_Hind3_2</td><td>CCCAAGCTTCGTAGACCACCCCGACAT</td><td>pGEM-T Easy-based intermediary construct</td></tr><tr><td>PBR325_CAT_AgeI</td><td>TACCGGTGTCCCTGTTGATAC</td><td>pBR325</td></tr><tr><td>PBR325_CAT_Stul</td><td>TAGGCCTGCCCTGAACCGAC</td><td>pBR325</td></tr><tr><td>ssrAKO_H1fwd</td><td>GTCGACTCTAGAGGATCCCCTGACGGCGGCCCCGGCCA</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>ssrAKO_H1_rev</td><td>TCTTTTCGTTAAAACCTTGACAGGTCGACGACCAGTCTACGGCAAAG</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>ssrAKO_H2_fwd</td><td>TCAAGGTTTTAACGAAAAGAGATAAGCCCC</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>ssrAKO_H2_rev</td><td>CCGAATTCGAGCTCGAGCCCGACTACCGAGCAGATCGG</td><td>P. aeruginosa</td></tr></table>
<table><tr><td></td><td></td><td>genomic DNA</td></tr><tr><td>SmpBKO_H1fwd</td><td>CCGAATTCGAGCTCGAGCCCAACGTCGTAACGCAGGAC</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>SmpBKO_H1_rev</td><td>AGGGACGCGCAGGCGGCGCATTATAGGG</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>SmpBKO_H2</td><td>TGCGCCGCCTGCGCGTCCCTGCCCCGCGC</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>SmpBKO_H2_rev</td><td>GTCGACTCTAGAGGATCCCCTTTACAAAAGCGAGCGCTCAG GTTAAGAGTCGAGCGTTCG</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>clpPKO_H1</td><td>GTCGACTCTAGAGGATCCCCTGATGTCGACAACATGCTC</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>clpPKO_H1_rev</td><td>TTGCGGAGCCGTCTTGCGATCACTCCCTAAC</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>clpPKO_H2</td><td>ATCGCAAGACGGCTCCGCAATACTGGCG</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>clpPKO_H2_rev</td><td>CCGAATTCGAGCTCGAGCCCCCGAACTTGACGAGATCTTCC</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>Bios1_H1</td><td>TGCAGGTCGACTCTAGAGGATCCCCAATGGGGGGAGGATGC</td><td>P. aeruginosa genomic DNA</td></tr></table>
<table><tr><td>Bios1_H1_rev</td><td>AAGCGGGCAGTGAGCCTGATCCTTTCGTCGG</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>Bios1_lacIq-Ptac_fwd</td><td>AAAGGATCAGGCTCACTGCCCCGCTTTCCAG</td><td>pMMB67EH</td></tr><tr><td>Bios1_lacIq-plac_rev</td><td>AGGTACCGAATTCGAGCTCGAGCCCTCTGTTTCCTGTGTGAAATTGTTATCC</td><td>pMMB67EH</td></tr><tr><td>Bios1_H1-lacIq-plac_rev</td><td>CCTGTCTCCTCGTCTGTTTCCTGTGTGAAATTGTTATCC</td><td>pBios1_INT</td></tr><tr><td>Synth_Bios1_mid-H2_fwd</td><td>CACAGGAAACAGACGAGGAGACAGGCATGAC</td><td>Synth_Bios1_mid-H2</td></tr><tr><td>Synth_Bios1_mid-H2_rev</td><td>AGGTACCGAATTCGAGCTCGAGCCCGTTGTGGCAGAGCACG GTC</td><td>Synth_Bios1_mid-H2</td></tr><tr><td>Bios2_fwd</td><td>GAG GAT CCC CAT GAC CGA GAA TGC CCT GCT GTT G</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>Bios2_hemH_trq_rev</td><td>TCA AAG CCG TCA TAC CGC GCC GCG CAG C</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>Bios2_hemH-tmRNAtag_fwd</td><td>ACCCCGCCTGGGTCGGCGCCCTGGGCGAGA</td><td>psynth_Bios2</td></tr><tr><td>Bios1_hemH-tmRNAtag_rev</td><td>AATGAGCACTTCAAAGCCGTCATACCGCGCCG</td><td>psynth_Bios2</td></tr><tr><td>Bios2_H2_fwd</td><td>GCG CGG TAT GAC GGC TTT GAA GTG CTC ATT TG</td><td>P. aeruginosa genomic DNA</td></tr><tr><td>Bios2_H2_rev</td><td>GCT CGA GCC CAG TTG TGG CAG AGC ACG G</td><td>P. aeruginosa</td></tr></table>
<table><tr><td></td><td></td><td>genomic DNA</td></tr><tr><td>pMMb67EH_w/o_p<br/>rom_fwd</td><td>GGCTGTTTTGGCGGATGAG</td><td>pMMB67E<br/>H</td></tr><tr><td>pMMb67EH_w/o_p<br/>rom_rev</td><td>GCGCAACGCAATTAATGTAAG</td><td>pMMB67E<br/>H</td></tr><tr><td>pBAD Prom Fwd</td><td>TTACATTAATTGCGTTGCGCTTTTTACGGTTCCTGGCC</td><td>pGLO</td></tr><tr><td>pBAD Prom ClpP_r<br/>rev</td><td>TGCGAGACATATGTATATCTCCTTCTTAAAGTTAAACAAAAT<br/>TATTTC</td><td>pGLO</td></tr><tr><td>ClpP_comp_fwd</td><td>AGATATACATATGTCTCGCAACTCTTTTATTCCGC</td><td>P.<br/>aeruginosa<br/>genomic<br/>DNA</td></tr><tr><td>ClpP_comp_rev</td><td>TCTCATCCGCCAAAACAGCCTTAGACGGCCAGGTCGCG</td><td>P.<br/>aeruginosa<br/>genomic<br/>DNA</td></tr><tr><td>pBAD Prom SmpB<br/>_rev</td><td>GCTTAGCCATATGTATATCTCCTTCTTAAAGTTAAACAAAAT<br/>TATTTC</td><td>pGLO</td></tr><tr><td>SmpB_comp_fwd</td><td>AGATATACATATGGCTAAGCAGAAGAAACACC</td><td>P.<br/>aeruginosa<br/>genomic<br/>DNA</td></tr><tr><td>SmpB_comp_rev</td><td>TCTCATCCGCCAAAACAGCCTCAGTCGTCCTTGCCGTG</td><td>P.<br/>aeruginosa<br/>genomic<br/>DNA</td></tr><tr><td>pBAD Prom For ss<br/>rA_rev</td><td>AATCCGCCGCTATGGAGAAACAGTAGAGAGTTG</td><td>pGLO</td></tr><tr><td>SSrA_comp_fwd</td><td>TTTCTCCATAGCGGCGGATTGGACTCTC</td><td>P.<br/>aeruginosa<br/>genomic<br/>DNA</td></tr><tr><td>SSrA_comp_rev</td><td>TCTCATCCGCCAAAACAGCCAAAAAGCCCCTGAAATTCGCT<br/>AAG</td><td>P.<br/>aeruginosa</td></tr></table>
genomic
DNA

## 2.2 Plasmids and strains construction (supplementary Fig. S1, Fig. S2 & Table S1)

To assess the viability of the *P. aeruginosa* *hemH* mutant and its capacity to produce red fluorescence, the central part of the gene was replaced with the gene coding for chloramphenicol acetyltransferase (CAT) from the pBR325 plasmid, to facilitate selection. The H1-*hemH*-H2 fragment was amplified from *P. aeruginosa* PA14 genomic DNA using the PA14_H1-*hemH*-H2_HindIII_1 and PA14_H1-*hemH*-H2_HindIII_2 primers, then cloned via TA cloning into the pGEM-T Easy system. Next, the *cat* cassette, amplified from pBR325 with the PBR325_CAT_AgeI and PBR325_CAT_StuI primers, was cloned into the pGEM-T Easy H1-*hemH*-H2 at the *AgeI* and *StuI* restriction sites. Finally, the H1-*hemH*::*cat*-H2 *HindIII* fragment was cloned into the pEXG2 plasmid at the *HindIII* site, creating pKO-hemH. All restriction enzymes used for this construction were purchased from New England Biolabs.

The pKO plasmid series, except for pKO-hemH, was constructed by cloning fragments flanking the gene to be deleted into the SmaI site of pEXG2. The fragments were obtained by PCR using Q5® DNA polymerase (New England Biolabs) and the primers listed in Table 1. The amplification products were purified using the Monarch® PCR & DNA Cleanup Kit (New England Biolabs), and the final constructs were obtained using HiFi assembly (New England Biolabs) and the NEBuilder® assembly tool, according to the manufacturer's instructions.

The pBios1-INT plasmid, which uses pEXG2 as a backbone, contains the upstream homology sequence of *hemH* (H1), obtained using the Bios1_H1 and Bios1_H1_rev primers with genomic DNA as the template. Downstream of this sequence, the plasmid includes the IPTG-inducible *lacIq*-Ptac-lacO promoter, derived from the pMMB67EH plasmid (Fürste et al., 1986), and amplified using the
Bios1_lacIq-Ptac forward and Bios1_lacIq-plac_rev primers. From pBios1-INT, the H1-lacIq-Ptac-lacO
fragment was amplified using the Bios1_H1 and Bios1_H1-lacIq-plac_rev primers and cloned into
pEXG2, upstream of the synthetic fragment present on psynth_Bios2 mid, amplified using the
Synth_Bios1 mid-H2 fward and Synth_Bios1 mid-H2 rev primers, creating the pBios1 plasmid (Table
1, Fig. S1).

The pBios2 plasmid was obtained by cloning most of the *hemH* gene, amplified using Bios2_fwd and Bios2_hemH_trq_rev, followed by a fragment containing the rest of the *hemH* gene fused with the tmRNA degradation tag, amplified from psynth_Bios2 using Bios2_hemH-tmRNAtag_fwd and Bios2_hemH-tmRNAtag_rev primers, and the downstream homology sequence of *hemH* (H2) amplified using the Bios2_H2_fwd and Bios2_H2_rev primers. The pBios2 uses the pEXG2 linearized by *Sma*I as backbone. (Table 1, Fig. S2)

The pcomp plasmid series has been created using the same supplies as the pKO series. The lactose-
inducible promotor and multiple cloning site of the pMMB67EH has been replaced by the arabinose
inducible promotor and AraC regulator of the pGLO plasmid. The RBS upstream of the promotor has
been conserved if necessary. For each complementation, the corresponding native gene has been cloned
downstream of this new promotor.

All mutant strains were obtained by following the method described in ²⁴ using the “puddle” variation
and by replacing LBNS medium by CAA. The sequences of the biosensor strains replacing the *hemH*
gene are compiled in table S1.

The inhibitor plasmids pantisens_ssrA_1 and pantisens_ssrA_2 have been created by cloning the
synthetic fragments from Genscript (Table S1), between MluI and PstI sites of the pMMB67EH using
Hi-T4 DNA Ligase (NEB).
## 2.3 High throughput compound testing procedure

For biosensor testing, *P. aeruginosa* strains were inoculated at an initial A<sub>600</sub> of 0.05 in Btm1 for Bios1 and Btm2 for Bios2. Tests were performed in 96 well flat bottom plates (ref: 353072, Falcon). The plate was incubated in the BioTek Synergy H1 Multimode Reader (Agilent) at 37 °C. Both the A<sub>600</sub> and the red fluorescence intensity (λ<sub>ex</sub> = 400nm/λ<sub>em</sub> = 620nm) were measured.

Data analysis is performed as follows: the A<sub>600</sub> is linearized using the formula log<sub>2</sub>(100 * A<sub>600</sub>). The normalized fluorescence intensity is calculated by dividing the fluorescence intensity (λ<sub>ex</sub> = 400nm / λ<sub>em</sub> = 620nm) by the linearized A<sub>600</sub>. The inflection point of each curve is then determined using the "gcplyr" R package to rephase the curves<sup>25</sup>. Mean and standard deviation calculations are performed after linearization, normalization, and rephasing. Statistical analysis was performed using a Kruskal-Wallis test followed by a Dunn post-hoc test, with Bonferroni correction, on the points between 150-350 minutes for Bios1 and between 150-520 minutes for Bios2.

For specificity testing of the biosensors, tobramycin, netilmicin, and ciprofloxacin were used at concentrations of 0.08 µg/mL, 0.17 µg/mL, and 0.02 µg/mL, respectively. These concentrations correspond to one-third of the minimum inhibitory concentration (MIC), determined as the lowest concentration at which no visible growth was observed in a 96-well microplate using MH medium for the *P. aeruginosa* Δ*ssrA* mutant strain (data not shown).

For fluorescence intensity measurements on the lysate, the cell pellet was disrupted using a mixture of 0.1 M NH<sub>4</sub>OH and acetone in a 9:1 (v/v) ratio.

## 2.4 Mass spectrometry analysis

96 wells plates were inoculated in the previously stated conditions with either wild type or Bios1 *P. aeruginosa* strains. After 250 minutes of incubation, the cells were collected by centrifugation and
subsequently lysed by vortexing in 6 M formic acid. The samples were freeze-dried and then resuspended
in either acetone plus 1 % (v/v) formic acid or DMSO. The HPLC system used was an UltiMate 3000
HPLC RSLC cap system coupled to a QExactive mass spectrometer, equipped with a HESI II source
(Thermo Fisher Scientific, Bremen, Germany). Samples were acquired either in direct infusion mass
spectrometry (DI-MS) at 5 µL/min with a syringe pump or liquid chromatography coupled with mass
spectrometry (LC-MS), after loading on a trap column of 1 µL. Mass spectra (MS1) or tandem mass
spectra (MS2) were recorded at a resolution of 140,000 at m/z 200.
# 3 Results

## 3.1 Red fluorescence of P. aeruginosa Δ*hemH*

To develop a cell-based system for monitoring *trans*-translation, we leveraged the natural red fluorescence of the *P. aeruginosa* Δ*hemH* mutant due to the accumulation of protoporphyrin IX (PPIX), the substrate of ferrochelatase encoded by the *hemH* gene. To enhance PPIX production and bypass the regulation of the PPIX biosynthesis pathway (which occurs primarily at the *hemA* gene encoding the glutamyl-tRNA reductase), we supplemented the growth medium with 5-aminolevulinic acid (ALA). This is a precursor in the tetrapyrrole biosynthetic pathway upstream of PPIX and downstream of glutamate-1-semialdehyde, the product of glutamyl-tRNA reductase (Fig. S3)²⁶,²⁷. The fluorescence intensity of the Δ*hemH* mutant is directly correlated with the concentration of ALA in the medium, confirming the role of ALA in modulating the signal of red fluorescence (Fig. S4).

To investigate the *trans*-translation process in *P. aeruginosa*, we engineered two distinct biosensor strains. “Biosensor 1” (Bios1) was designed to assess the effect of molecules on the tagging efficiency of *trans*-translation mediated by the SmpB-tmRNA system, while “Biosensor 2” (Bios2) serves as a control to access the impact of the tested molecules on the degradation of tmRNA-tagged proteins by the ClpP1-containing protease complex, or their effect on PPIX synthesis. Both strains are based on modifications of the *hemH* gene which encodes ferrochelatase, the catalyst for inserting ferrous iron into protoporphyrin IX.

## 3.2 Bios1: A tool for measuring *trans*-translational activity

To specifically induce red fluorescence under conditions when *trans*-translation is not inhibited, we modified the *hemH* gene to produce a protein that becomes a substrate for tmRNA-SmpB-mediated tagging. To do this, we replaced the native *hemH* gene by a modified version (*hemH*-ns) lacking the
translation termination codon (Fig. S1). To ensure tight regulation, we then placed this modified gene
under the control of the IPTG-inducible *lacIq-Ptac-lacO* promoter derived from the pMMB67EH
vector<sup>22</sup>. To guarantee transcription termination, the non-STOP gene is followed by a sequence
containing two Rho-independent 5S ribosomal RNA (PA14_55620)<sup>28</sup>. The genetic construct also
ensures the absence of a stop codon for translation, regardless of the reading frame (see Materials and
Methods). The resulting stalled ribosome complex at the end of the *hemH-ns* mRNA then requires *trans*-
translation. If *trans*-translation is functional in the Bios1, the tagged ferrochelatase is sent for degradation
by the ClpP1-containing proteases such as ClpXP1<sup>29</sup>, PPIX accumulates, and a red fluorescent signal is
emitted (Fig. 1). A decrease in red fluorescence will indicate an inhibition of *trans*-translation.
<figure><img src="image_2.png" /></figure>
**Figure 1.** Schematic of Bios1 functionality. Upon induction, the synthetic construct generates a non-STOP mRNA, which must be resolved by *trans*-translation. This rescue process tags the newly synthesized HemH protein for degradation by adding a tm-RNA-TAG ("ssrA-TAG") at the C-terminus. Ferrochelatase degradation leads to the accumulation of protoporphyrin IX, which results in red fluorescence within the bacterial cell. If *trans*-translation is absent, inhibited, or overwhelmed, the HemH protein remains untagged and free. As a result, protoporphyrin IX can be converted into heme, reducing the red fluorescence.

To optimize the culture conditions for Bios1, we tested the concentrations of three key compounds: iron, a substrate for ferrochelatase in the production of heme from PPIX; aminolevulinate, a precursor in the heme biosynthetic pathway, and IPTG, an inducer of the *hemH*-ns gene. To effectively control iron concentrations, we used CAA medium as the base. We then evaluated the performance of each tested combination by measuring and comparing the fluorescence signals at the 250-minute mark in both Bios1 and its Δ*ssrA* mutant strain. We were looking for a high fluorescence in Bios1, with a significant difference between Bios1 and its Δ*ssrA* mutant strain. The optimal combination is 10 µM IPTG, 0.5 mM ALA, and 40 µM FeSO₄ (Fig. 2).
<figure><img src="image_3.png" /></figure>

**Figure 2.** The Difference in red fluorescence (λex = 400 nm/λem = 620 nm) normalized by absorbance (600 nm) between Bios1 and its Δ*ssrA* mutant strains at the 250-minute mark illustrates the maximum stable signal under the tested growth conditions. Data is only shown for [FeSO₄] = 40 µM, as this parameter did not influence fluorescence intensity. The colors indicate the concentration of aminolevulinate, and the shapes indicate the concentration of IPTG. Three technical repeats over four biological repeats were used for mean calculation.

To validate the correlation between fluorescence levels and *trans*-translation, we constructed Bios1 Δ*smpB* and Bios1 Δ*ssrA* strains, in which *trans*-translation is disrupted. In the wild-type (WT) background, tmRNA and SmpB are active, so PPIX accumulates. In contrast, in the Δ*smpB* or Δ*ssrA* mutant strains, we expected a reduction in red fluorescence because the untagged HemH is not targeted
for degradation. Thus, PPIX converts to heme, reducing the accumulation that causes red fluorescence (Fig. 1). It must be noted that the Δ*ssrA* and Δ*smpB* mutant strains of *P. aeruginosa*, in both wild-type and biosensor backgrounds, exhibit minimal reductions in growth rates (Figs. 3A, B). This observation suggests that disrupting *trans*-translation has a limited impact on cell growth, likely due to the activity of alternative rescue pathways, *e.g.* ArfA and ArfB. Therefore, observed differences of fluorescence cannot be attributed to variations in cell growth rates or changes in cell morphology. Such resilience is a necessary condition for the development of an *in vivo* system for screening. To ensure that the decreased red fluorescence we observed in the mutated *Bios1* strains was solely due to the deletion of *ssrA* or *smpB*, we performed complementation assays using specially designed plasmids carrying the missing gene under the control of an arabinose-inducible promoter. Complementation of the *Bios1* Δ*ssrA* strain fully restored the *Bios1* phenotype, while complementation of the Δ*smpB* strain only partially restored it. This partial restoration is probably due to the reduced growth rate caused by the metabolic burden of the pcomp-smpB plasmid, as observed in both the *Bios1* and *Bios1* Δ*smpB* complementation strains (Fig. 3).
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Validation of the Bios1. (A-B) Growth curves of Bios1, the Bios1 Δ*ssrA* (A) or Δ*smpB* (B) mutant strain, and the corresponding complemented strains. (C-D) Fluorescence (λex = 400nm/ λem = 620nm) normalized by absorbance (600 nm) of Bios1, its Δ*ssrA* (C) or Δ*smpB* (D) mutant strain, and the corresponding complemented strains. Three technical repeats were used for mean calculation and standard deviation (Kruskal-Wallis then Dunn test; **, p < 0.01, ***, p < 0.001).</figcaption></figure>

The conversion of ALA to PPIX is a multistep process that involves the synthesis of various porphyrins, which are organic compounds known for their ability to emit red fluorescence under specific
conditions. In *P. aeruginosa*, the heme biosynthetic pathway can split into two branches to produce
several porphyrin derivatives, including PPIX and coproporphyrin III, all of which can contribute to red
fluorescence³⁰. To ensure therefore that the red fluorescence we observed in *Bios1* was predominantly
due to PPIX accumulation, we turned to mass spectrometry analysis. Both direct infusion mass
spectrometry (DI-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to detect,
quantify relatively, and compare the amounts of porphyrins present in WT and *Bios1* *P. aeruginosa*
strains. These tests confirmed the presence of both PPIX and coproporphyrins (Fig. 4).

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Ratio of protoporphyrin IX (PPIX) and coproporphyrin (CP) extracted from the *Bios1* and the wild-type (WT). (A) ratio of the [X+H]⁺ species with DMSO as solvent. (B) ratio of the [X+2H]⁺ species with DMSO as solvent. (C) ratio of the [X+H]⁺ species with acetone + 1 % formic acid as solvent. (A)</figcaption></figure>
and (B) were obtained by DI-MS and (C) by LC-MS. (Kruskal-Wallis test followed with Dunn test; **, p-value <0.01; *, p-value <0.06).

The ratios of PPIX to coproporphyrins observed in these experiments support the hypothesis that protoporphyrin IX accumulates in the *P. aeruginosa* Bios1 strain, and that it contributes significantly to the red fluorescence detected there (Fig. 4). This accumulation is consistent with the conversion of aminolevulinate into mainly PPIX as well as with the role of HemH in converting the PPIX into heme.

### 3.3 Bios2: A tool to measure the proteolytic activity of ClpP1-containing protease

We developed Bios2 as a control to verify that the molecules inhibiting Bios1 fluorescence do not interfere with ClpP1-mediated proteolysis of tmRNA-tagged proteins or with PPIX production by disrupting the heme biosynthesis pathway or any other canonical translation process other than *trans*-translation. *P. aeruginosa* has two ClpP peptidase isoforms, ClpP1 and ClpP2. The *clpP1* gene is expressed throughout the growth phases, while the *clpP2* gene is regulated by quorum-sensing signaling molecules and the transcription factor LasR. Their respective roles in relation to *trans*-translation are not well understood. However, ClpP1 is known to be an essential component of the ClpXP enzyme which recognizes and degrades tmRNA-tagged translation products. In order for ClpP2 to function as a protease, it must co-assemble with ClpP1 to form a heterocomplex, and only the ClpP1 ring of that complex can bind ClpX³¹. Therefore, the *clpP1* coding gene was chosen as a target to construct the negative control.

To construct Bios2, the native *hemH* gene in *P. aeruginosa* PA14 was replaced with the *hemH* gene in translational fusion with the tmRNA-tag of the WT strain (ANDDNYALAA*). When proteolysis mediated by the ClpP1-containing complex is effective, HemH fused with the tmRNA-degradation tag
is degraded, and this leads to an accumulation of PPIX in the cell and thus increased red fluorescence (Fig. 5). To see if this fluorescence could be linked with proteolytic activity, we deleted the *clpP1* gene in Bios2. While the Bios2 shows increased fluorescence over time, the Δ*clpP1* mutant does not, and this is consistent with the previously established role of ClpP1 in the degradation of GFP-tmRNA-tagged proteins even in the presence of ClpP2³⁰. The reintroduction of a *trans*-encoded *clpP1* partially restored the Bios2 phenotype, although with limited efficiency (Fig. 6). The reduced growth observed during complementation assays suggests that overexpression of the pleiotropic ClpP1 protease impacts bacterial metabolism, although the growth rate is not significantly affected (Fig. 6). This validation confirmed that Bios2, which contains the complex with ClpP1 peptidase, produces red fluorescence due to the degradation of the HemH-tmRNA-tag fusion protein. The fluorescence is significantly lower in Bios2 Δ*clpP1*, and similar results should be observed in the case exogenous inhibition of ClpP1-containing protease or PPIX synthesis.
<figure><img src="image_6.png" /><figcaption>**Figure 5.** Schematic of Bios2 functionality. Upon induction with IPTG, the synthetic construct expresses HemH fused with the tmRNA degradation tag. The tagged HemH is sent to be degraded by the ClpP1-associated protease complex, PPIX accumulates, and emits red fluorescence. If ClpP1-mediated proteolysis is inhibited or absent, the tagged HemH persists, which allows heme synthesis and thus</figcaption></figure>
decreases fluorescence. Furthermore, molecules that inhibit the heme biosynthesis pathway upstream of
HemH will also lead to reduced red fluorescence in Bios2.

<figure><img src="image_7.png" /></figure>

**Figure 6.** Validation of Bios2. (A) Growth curve of Bios2, its *ΔclpP1* mutant strain, and the corresponding complemented strains. (B) Fluorescence (λex = 400nm/ λem = 620nm) normalized by absorbance (600 nm) over time for Bios2, its *ΔclpP1* mutant strain, and the corresponding complemented strains. Three repeats were used for mean calculation and standard deviation (Kruskal-Wallis then Dunn test; ***, *p* < 0.001).
## 3.4 Effect of inhibitors on the biosensor system

To evaluate the system's responses under various conditions, we tested its performance in the presence of commonly used antibiotics as well as specifically designed inhibitors.

### The effects of common antibiotics on biosensor performance

To ensure the specificity of these biosensors developed for detecting *trans*-translation inhibitors, it was essential that we evaluate their performance in the presence of commonly used antibiotics. This helps rule out any potential false positives caused by antibiotic interference with the fluorescence output of the biosensors. We chose three antibiotics for testing: the ribosomal-targeting drugs netilmicin and tobramycin, and ciprofloxacin, which targets DNA gyrase, a non-ribosomal site. Each antibiotic was administered at one-third of its minimum inhibitory concentration (MIC) to assess whether sub-inhibitory levels would affect the biosensor fluorescence. The results demonstrated that none of these antibiotics significantly altered the fluorescence levels in either biosensor. This suggests that these biosensors are robust and that they maintain their specificity even in the presence of common antibiotics (Fig.S5).

## 3.5 The effects of specifically designed inhibitors

To test the sensitivity of our detection system, we investigated the impacts of specifically designed *trans*-translation inhibitors on the biosensors. Since no molecules specifically targeting *P. aeruginosa* *trans*-translation have been formally identified, we designed antisense RNAs as inhibitors. These inhibitors have both already been shown to affect *trans*-translation in *E. coli*³². The antisense RNAs, encoded by plasmids *pAntisens_1* and *pAntisens_2*, target the tmRNA MLD to inhibit its role in *trans*-translation. The *pAntisens_2* construct, enhanced with an *E. coli* *MicC* sRNA scaffold, was designed to improve the HFQ-mediated stability of the sRNA-tmRNA complex, thereby increasing its inhibitory
effect. We then measured the fluorescence from the biosensor strains as a quantitative assessment of *trans*-translation activity. In fact, the pAntisens_2 construct significantly reduced Bios1 fluorescence, indicating that *trans*-translation had been successfully inhibited, and the biosensor's inhibition specificity was confirmed when this reduction was not observed in Bios2. In contrast, the pAntisens_1 construct, lacking the MicC scaffold, showed a weaker, non-significant inhibitory effect, which highlights the importance of RNA stability in effective inhibition (Fig. 7).

<figure><img src="image_8.png" /><figcaption>**Figure 7.** Effect of *trans*-translation inhibitors on the biosensors. (A-B) Growth curves of Bios1 (A) and Bios2 (B). (C, D) Fluorescence (λex = 400nm/ λem = 620nm) normalized by absorbance (600 nm) over</figcaption></figure>
time of Bios1 (C) and Bios2 (D). Three repeats were used for mean calculation and standard deviation
(Kruskal-Wallis then Dunn test; ***, * p < 0.001).

**3.6 Discussion**

We report the development of two biosensor strains that can detect novel anti-*trans*-translation
compounds targeting *P. aeruginosa* PA14. This system is specifically designed to identify compounds
that target *trans*-translation. The deletion induces various phenotypes in bacteria, including a loss of
virulence, a decrease in antibiotic tolerance, and more generally, a reduction in stress resistance and the
resuscitation of persister cells⁷. The *trans*-translation system therefore constitutes a target of interest for
combating infections, especially since this system is absent in eukaryotes. To date, there has been no *in
vivo* system for measuring *trans*-translation in this strain. The system we developed is particularly suited
to the constraints associated with *P. aeruginosa* (intrinsic fluorescence, resistance to many antibiotics)
and offers a completely innovative approach compared to the *E. coli*-based system previously developed
³². By integrating the genetic system into the genome, we ensure stability and eliminate biases associated
with plasmid-based systems.

These biosensors are based on the specific degradation of the ferrochelatase HemH, which converts
red fluorescent protoporphyrin IX into non-fluorescent heme. Bios1 effectively identifies *trans*-translation inhibition. Bios2 serves as a control to confirm that observed effects result from targeted inhibition rather than general cellular disruptions (Fig. S6) Incorporating an IPTG-inducible promoter upstream of the *hemH* gene decouples its expression from endogenous regulatory mechanisms, while the addition of aminolevulinate bypasses the upstream regulation of *hemA*, further enhancing system specificity. This whole-cell-based system has the advantage of targeting active molecules capable of
penetrating *P. aeruginosa* cells. In addition, in an improvement over an acellular system, the system adjusts for the capacity of efflux systems to expel these molecules. Under our testing conditions, *trans*-translation mutant strains exhibit no significant growth impairment, suggesting that these alternative rescue factors are sufficiently efficient to resolve non-stop ribosome complexes when *trans*-translation is disrupted. The miniaturized 140-µL microplate format enables automated kinetic fluorescence monitoring, making it cost-effective and suitable for high-throughput screening of antimicrobial candidates. The limitations of this system are related to the potential influence of certain molecules and environmental factors on both *trans*-translation and porphyrin synthesis or proteolysis, which may lead to their elimination from the selection based on their effects on Bios2 (Fig. S6). This limitation is partially mitigated by the standardization of the medium and the strict control and monitoring of experimental conditions as part of the quality control process.

Another advantage of this system's working principle is that it can be applied to other bacteria in the ESKAPEE group, including *Staphylococcus aureus*, *Acinetobacter baumannii*, *Klebsiella pneumoniae*, and *E. coli*. It has already been shown that these bacteria produce red fluorescence when exposed to violet light during fluorescence imaging³³. Provided that genetic tools are available to modify the genome of target strains, using a genomically integrated system instead of a plasmid-based one eliminates the need for antibiotic resistance for system maintenance. Furthermore, this *hemH*-based system does not require a dedicated insertion site, and it leverages the intrinsic metabolic processes of the bacteria. Adaptation to other bacterial species will depend on their ability to take up exogenous heme to compensate for the auxotrophy caused by *hemH* modification. Additionally, they must be capable of growing under controlled conditions that are favorable to red fluorescence emission.
Our next objective will be to select molecules at concentrations having no significant effect on biosensor growth and which decrease Bios1 fluorescence without affecting that of Bios2. This differential effect will help us to identify inhibitors specifically targeting *trans*-translation rather than ClpP1 activity or general metabolic pathways. These new biosensors are a versatile and efficient tool for identifying novel antimicrobial agents, can be applied over a broad range of bacterial species, and have the potential to accelerate the discovery of targeted therapies against critical drug-resistant pathogens.
**Supporting Information**

Figures S1 & S2: construction of the pBios1 and pBios2

Figures S3: Schematics of porphyrin metabolism pathway of *P. aeruginosa* UCBPP-PA14

Figure S4: Estimation of the relative PPIX levels in lysates of *P. aeruginosa* WT and *ΔhemH* based on fluorescence measurements calibrated with a PPIX standard curve.

Figures S5: Effect of commonly used antibiotics at a concentration of MIC/3 on the biosensors.

Figure S6: Schematic of the Biosensor System Workflow. Molecule A specifically targets *trans*-translation, affecting only Bios1, whereas Molecule B impacts both biosensors.

Table S1: Biosensors sequences

Table S2: Synthetic sequences used to create the inhibitor plasmids.

**4 Author Contributions**

BLH and CB developed the experimental strategies for Microbiology, while NLY and PJ designed the chemistry experiments. The experiments were conducted by BLH, TC, SGD, NLY, and PJ. The data analysis and conclusion drafting were carried out by BLH, CB, and RG. The manuscript was written by BLH, CB, and RG.

**5 Conflict of Interest**

The authors declare no competing financial interests

**Acknowledgments**
This work was supported by grant RF20220503018 from the French Cystic Fibrosis associations
Vaincre la Mucoviscidose and Association Grégory Lemarchal, as well as by the Fondation pour la
Recherche Médicale (Equipe FRM EQU202203014663) and the Agence Nationale de la Recherche
(ANR-23-CE18-0031-01).
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Developing Biosensors for Specific Assessment of *Trans*-translation in *Pseudomonas aeruginosa*

Bastien L’Hermitte<fnref n="1" />, Thomas Chauvet<fnref n="1" />, Sylvie Georgeault-Daguenet<fnref n="1" />, Nicolas Le Yondre<fnref n="2" />, Philippe Jehan<fnref n="2" />, Reynald Gillet<fnref n="1" />* and Christine Baysse<fnref n="1" />*

<footnote n="1">¹ University of Rennes, CNRS, Institut de Génétique et Développement de Rennes (IGDR) UMR6290, 35000 Rennes, France</footnote>

<footnote n="2">² University of Rennes, CNRS, Centre Régional de Mesures Physiques de l'Ouest (CRMPO) ScanMAT UAR 2025, 35000 Rennes, France</footnote>

<footnote>*Co-corresponding authors: reynald.gillet@univ-rennes.fr ; christine.baysse@univ-rennes.fr</footnote>
<figure><img src="image_9.png" /><figcaption>**Figure S1:** Schematic representation of the construction of the plasmid pBios1.</figcaption></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>Figure S3: Porphyrin metabolism pathway of *P. aeruginosa* UCBPP-PA14 from L-Glutamyl-tRNA to Protoheme adapted from pathway map pau00860¹.</figcaption></figure>

(1) Kanehisa, M.; Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. *Nucleic Acids Res.* **2000**, *28* (1), 27–30. https://doi.org/10.1093/nar/28.1.27.
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>Figure S4: (A) Estimation of the relative PPIX levels in lysates of *P. aeruginosa* WT and *ΔhemH* (B) based on fluorescence measurements calibrated with a PPIX standard curve.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>Figure S5: Effect of commonly used antibiotics at a concentration of MIC/3 on the biosensors. (A-B) Growth curves of Bios1 (A) and Bios2 (B). (C, D) Fluorescence (λex = 400nm/ λem = 620nm) normalized by absorbance (600 nm) over time of Bios1 (C) and Bios2 (D). Three repeats were used for mean calculation and standard deviation (Kruskal-Wallis then Dunn test; ***, p < 0.001).</figcaption></figure>

<figure><img src="image_15.png" /></figure>
Figure S6: Schematic of the Biosensor System Workflow. Molecule A specifically targets *trans*-translation, affecting only *Bios1*, whereas Molecule B impacts both biosensors. This schematic illustrates the selection process for cases where a molecule demonstrates an effect on at least *Bios1*.
<table><caption>**Table S1: Biosensors sequences**</caption><thead><tr><th>Name</th><th>DNA sequence</th></tr></thead><tbody><tr><td>Bios1</td><td>TCACTGCCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGG GAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGA TTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGTGGTTTGCCCCAGCAGGCGA AAATCCTGTTTGATGGTGGTTAACGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCAC TACGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTT ATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGGATTTGCT GGTGACCCAATGCGACCAGATGCTCCACGCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTG TTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTCAGGCAGCTTCCACAGC AATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGT GCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGGTCTACCATCGACACCACCACGCTGGCACCCAGT TGATCGGCGGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGC AACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCT CCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGG GAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCAC CACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCACCATTCGATGG TGTCAACGTAAATGCCGCTTCGCCTTCGCGCGCGAATTGCAAGCTGATCCGGGCTTATCGACTGCACG GTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCA CTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAAC GGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTG TGAGCGGATAACAATTTTcacacaggaacagaCGAGGAGACAGGCATGACCGAGAATGCCCTGCTGTT GCTCAACCTGGGCTCCCGGATTCGACCCGGGTGGAGGATGTCCGCCGCTATCTCGACCAGTTCCTCA TGGATCCCTACGTGGTCGACCTGCCCTGGCCGCTGCGGCGCCTGCTGGTGTCGCTGATCCTGATCAAG CGTCCGGCCGAATCGGCGCACGCCTACAGCTCGATCTGGTGGGACGAAGGGTCGCCACTGATCGTCCT CAGTCGTCGCCTGCAGGAAGCGATGAAGCCGCACTGGCCGCATGGACCGGTGAACTGCGGATGCGCT ACGGCCAGCCGGCCATCGAGAAGGTCCTGCTCGACCTGGCGCGCCGCGGTATCCGCGGGTGACCCTG GCGCCGCTGTATCCACAGTTCGCCGACAGCACCACGACCACCGCCGAACAAGAGGTGCGCCGGGTGAT CGCCGCCCATCGCCTGGAGCTGGAGGTCAGCACGCTGCCGCCGTTCTACGACCAGCCGGTCTACCTCG ATGCCCTGGTGGAAAGCGTGCGTCCGTACCTGCAGCAGCCCCACGACCATCTGTTGCTGAGCTTCCAT GGCTTGCCCGAGCGGCACATCCGCAAGCTGGTGAAGGATCCTGCCACGACCTGCTGGCCGAGAACAG CCGAACGTCAGCCCCGAAGCCCTCGCCCTCTGCTACCGCAGCCAGTGCCCTGCGCACCGCCGAGGCCT TCGCCGAGCGCGCCGGCCTGGAGCAGGGGCGCTGGTCGGTGTTCGTTCAGTCGCGCCTGGGGCGCGCC AAGTGGATCGAGCCCTATACCGACGCCAAGCTCGACGAACTGGTCAGCGTGGGGTGAAGCGCCTGCT GGTGATGTGCCCCGGCGTTTCGTCGCCGACTGCATCGAGACTCTGGAGGAAATCGGCATGCGCGCCGCG AGCAGTTCATCAGCGCCGGCGCGAGGACCTGGTGCTGATTCCCTGCCTCAACGACCACCCCGCCTGG GTCGGCGCCCTGGGCGAGATGAGCGGGCGCTTGGCCAGGCCGCTCCAAAACCCCTGGTCAGCGATGAC CAGGGGTTTTGCTTTTTCGGCGCGGAAAAAGGCGTGCTTCCTGTTCACCTTGCCGAACCCCTCCTGGCA TGTCCCTTTCGCGTCTAGCGTAGCGAGAGTCCGGGCGGATCCGCATGACGCTGGCGCTCTTGCGTTC GCCGTACCGATCCCGATCCGCGAGTTAGAGCCTCGCTTCGAGGAGAAAGACAAAAGATGGCTTCCGGT GAAGGGAATCGCTTTTACTGTATTATAACAGTATCTTGAGCGCTCCCCCCTTCGTGGCCATCC CTGTTGGCCATCCCAAGGCAGCTCGTCTCTCGAGCTGCCTTTTTCTTTGTC</td></tr><tr><td>Bios2</td><td>TCACTGCCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGG GAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGA TTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGTGGTTTGCCCCAGCAGGCGA AAATCCTGTTTGATGGTGGTTAACGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCAC TACGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCAGCGCCATCT GATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTT ATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCGCTAACAGCGCGATTTGCT GGTGACCCAATGCGACCAGATGCTCCACGCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTG</td></tr></tbody></table>
<table>
  <tr>
    <td>TTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGC AATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGT GCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGT TGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGC AACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCT CCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGG GAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCAC CACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCACCATTTCGATGG TGTCAACGTAAATGCCGCTTCGCCTTCGCGCGCGAATTGCAAGCTGATCCGGGCTTATCGACTGCACG GTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCA CTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTTGCGCCGACATCATAAC GGTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTG TGAGCGGATAACAATTTCACACAGGAAACAGACGAGGAGACAGGCATGACCGAGAATGCCCTGCTGTT GCTCAACCTGGGCTCCCGGATTCGACCCGGGTGGAGGATGTCCGCCGCTATCTCGACCAGTTCCTCA TGGATCCCTACGTGGTCGACCTGCCCTGGCCGCTGCGGCGCCTGCTGGTGTCGCTGATCCTGATCAAG CGTCCGGCCGAATCGGCGCACGCCTACAGCTCGATCTGGTGGGACGAAGGGTCGCCACTGATCGTCCT CAGTCGTCGCCTGCAGGAAGCGATGAAGCCGCACTGGCCGCATGGACCGGTGGAACTGGCGATGCGCT ACGGCCAGCCGGCCATCGAGAAGGTCCTGCTCGACCTGGCGCGCCGCGGTATCCGGCGGGTGACCCTG GCGCCGCTGTATCCACAGTTCGCCGACAGCACCACGACCACCGCCGAACAAGAGGTGCGCCGGGTGAT CGCCGCCATCGCCTGGAGCTGGAGGTCAGCACGCTGCCGCCGTTCTACGACCAGCCGGTCTACCTCG ATGCCCTGGTGGAAAGCGTGCGTCCGTACCTGCAGCAGCCCCACGACCATCTGTTGCTGAGCTTCCAT GGCTTGCCCGAGCGGCACATCCGCAAGCTGGTGAAGGATCCTGCCACGACCTGCTGGCCGAGAACAG CCGAACGTCAGCCCCGAAGCCCTCGCCCTCTGCTACCGCAGCCAGTGCCCTGCGCACCGCCGAGGCCT TCGCCGAGCGCGCCGGCCTGGAGCAGGGGCGCTGGTCGGTGTTCGTCCAGTCGCGCCTGGGGCGCCGC AAGTGGATCGAGCCCTATACCGACGCCAAGCTCGACGAACTGGTGCAGCGTGGGGTGAAGCGCCTGCT GGTGATGTGCCCGGCGTTCGTCGCCGACTGCATCGAGACTCTGGAGGAAATCGGCATGCGCGGCCGCG AGCAGTTCATCAGCGCCGGCGGCGAGGACCTGGTGCTGATTCCCTGCCTCAACGACCACCCCGCCTGG GTCGGCCCTGGGCGAGATGAGCGGGCGCTTGGCCAGGCCGCTCGCCAACGACGACAACTACGCTCT AGCTGCTTAA</td>
  </tr>
</table>
<table><caption>**Table S2:** Synthetic sequences used to create the inhibitor plasmids.</caption><thead><tr><th>Name</th><th>DNA sequence<br>(antisense RNA)</th></tr></thead><tbody><tr><td>Antisens_1</td><td>GAATTCACGCGTTTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCC TAACTGCTGGCGGAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATT GCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTT AATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATT GGCAAATATTGACGGCCAGTTAAGCCATTTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAA TTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCC CGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCCAGAGAAGAAACCAATTGTCCATA TTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAATCGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCA CATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATATTAAGCAGCTAGAGCGTAGTTGTCGTCGTTGGCGTCGAC (GACGCUUUUUAAUCGCAACUCUCUACUGUUUCUCCAUAUUAAAGCAGCUAGAGCGUAGUUUGUCGUCGUUGGC)</td></tr><tr><td>Antisens_2</td><td>ACGCGTTTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTC TGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGC TTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATTATTGACGGCCAGTTAAGCCATTTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGTCGATAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAATCGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGA TTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATATTAAGCAGCTAGAGCGTAGTTGTCGTCGTTGGCTTTCTGTTGGGCCATTGCATTGCCACTGATTTTCCAACATATAAAAAGACAAGCCCGAACAGTCGTCCGGGCTTTTTTTTTT (AGCAGCUAGAGCGUAGUUUGUCGUCGUUGGCUUUUCUGUUUGGGCCAUUGCAUUGCCACUGAUUUUCCAACAUAUAAAAA GACAAGCCCGAACAGUCGUCGGGCUUUUUUUUUU)</td></tr></tbody></table>