Highly tunable TetR-dependent target gene expression in the acetic acid bacterium Gluconobacter oxydans

Abstract For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter ParaBAD. In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-ParaBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3’ region of the Ptet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. Key Points • A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11473-x.


Introduction
The acetic acid bacterium (AAB) Gluconobacter oxydans harbors the beneficial ability of regio-and stereoselective incomplete oxidation of a variety of substrates (e.g., sugars and sugar alcohols) in the periplasm by membrane-bound dehydrogenases (mDHs) and release of resulting products into the cultivation medium (Mamlouk and Gullo 2013;Mientus et al. 2017;Pappenberger and Hohmann 2014). Because of this feature, G. oxydans is industrially used for oxidative biotransformations of carbohydrates to produce e.g. the vitamin C precursor l-sorbose, the tanning lotion additive dihydroxyacetone, and 6-amino-l-sorbose used for the production of the antidiabetic drug miglitol (Ameyama et al. 1981;Gupta et al. 2001;Hekmat et al. 2003;Saito et al. 1997;Tkac et al. 2001;Wang et al. 2016).
For the expression of target genes in G. oxydans, so far only constitutive promoters have been used due to the lack of a regulatable promoter demonstrated to be functional and tunable in G. oxydans (reviewed in Fricke et al. 2021). Only recently the first tight system became available for tunable induction of gene expression in G. oxydans. This system is based on AraC-P araBAD and the induction by l-arabinose binding to the regulator protein AraC (Fricke et al. 2020). AraC typically represses the target promoter P araBAD by DNA bending in the absence of the inducer l-arabinose and activates it in the presence of the inducer by a modified binding to the promoter DNA and thereby releasing the bending (Schleif 2010). However, in G. oxydans, P araBAD is almost not active in the absence of araC and thus, repression by AraC is not required in G. oxydans for tightness of P araBAD in the absence of the inducer (Fricke et al. 2020). In contrast, in Gluconacetobacter and Komagataeibacter plasmid-based AraC-P araBAD was reported to be very leaky (Teh et al. 2019). While AraC typically is acting both as repressor and activator, the transcriptional regulators TetR and LacI only exert a repressor function and dissociate from their operator DNA when forming a complex with their respective inducer (Hillen et al. 1983;Khoury et al. 1991;Miller 1970;Sellitti et al. 1987;Wray and Reznikoff 1983). P tet and P lac then enable transcription of the downstream gene by RNA polymerase. In AAB both TetR-and LacI-dependent target gene expression have so far been reported to be very leaky or inhomogeneously induced. In Komagataeibacter rhaeticus iGEM, the TetR-P tet system from transposon Tn10 exhibited only approximately 1.5-fold induction due to high leakiness in the absence of the inducer anhydrotetracycline (Florea et al. 2016). With a LacI-based expression system in K. xylinus, the induction ratio also appeared to be low and only a fraction of cells showed some induction (Liu et al. 2020). For another LacI-based system, expression was also found to be very leaky in G. oxydans (Condon et al. 1991).
In this study, we aimed to test TetR-and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used recently for the construction of the araC-P araBAD system (Fricke et al. 2020). With pBBR1MCS-5-based plasmid constructs, we found always some leakiness of the LacI-P lacuv5 system, yet extremely tight and optimally tunable target gene expression with TetR-P tet making the latter system highly suitable for applications in G. oxydans and possibly in other AAB.

Recombinant DNA work
All DNA oligonucleotides used for the construction of plasmids and sequencing are listed in Table S1 and were synthesized by Eurofins MWG. All restriction enzymes were purchased from Thermo Scientific. Polymerase chain reaction (PCR), restriction, and ligation reactions for DNA manipulations were conducted according to standard protocols (Sambrook et al. 1989). DNA fragments were amplified using Q5 polymerases according to the conditions recommended by the manufacturer (New England Biolabs). Unless stated otherwise for the construction of all reporter plasmids, amplified DNA fragments were integrated in the restricted broad-host vector pBBR1MCS-5 in a one-step isothermal Gibson assembly (50 °C, 1 h) (Gibson et al. 2009). All cloning steps to obtain desired plasmids were conducted in E. coli S17-1 and plasmids were isolated using a QIAprep spin miniprep kit (Qiagen). Inserts of all constructed plasmids were checked for correctness by DNA sequencing (Eurofins MWG).

Construction of plasmids
For the construction of plasmids, the empty vector pBBR1MCS-5-T gdhM -MCS-T 0028 was generated from pBBR1MCS-5. It carries the terminator sequences of GOX0265 (T gdhM ) and GOX0028 (T 0028 ) flanking the multiple cloning site (MCS) to minimize interfering effects between the plasmid backbone and expression of the inserted genes.
P l a s m i d p B B R 1 M C S -5 -T gdhMtet R -P tet -m N G -T BBa_B1002 -T 0028 was constructed using the primer pair Derivative of pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 with ribosomal binding site AGG AGA (Hentschel et al. 2013)

Measurements of mNG fluorescence
Expression of promoter-reporter constructs in G. oxydans was monitored using the fluorescence protein mNeonGreen (mNG) as a reporter (Shaner et al. 2013). In shake flask experiments, the inducibility of P tet -derived mNG expression in G. oxydans was tested by the addition of 200 ng mL −1 anhydrotetracycline (ATc) from a 0.2 mg mL −1 stock solution in 50% ethanol. Non-induced reference cultures were supplemented with an equal volume of 50% ethanol. For experiments with P lacUV5 -controlled mNG expression, 1 mM of isopropyl-β-d-1-thiogalactopyranoside (IPTG) was supplemented from a 100 mM stock solution in water. An equal volume of water was added to non-induced reference cultures. Throughout the cultivation samples were taken to monitor growth (OD 600 ) by a spectrophotometer (UV-1800, Shimadzu) and fluorescence emission using an Infinite M1000 PRO Tecan reader (λ ex 504 nm / λ em 517 nm, gain 60, ex/em bandwidth 5 nm, infinite M1000 PRO Tecan). Using a BioLector system, 800 µL batches of d-mannitol medium were inoculated from overnight starter cultures to an initial OD 600 of 0.3 and incubated at 30 °C (1,200 rpm; 85% humidity) using 48-well Flowerplates® (m2p-labs). Backscattering light intensity (A 620 nm ) for growth and fluorescence emission (λ ex 510 nm / λ em 532 nm) were monitored online during the cultivation. Backscatter and fluorescence signals were measured using gain 15 or 20 and 50 or 60, respectively, as indicated in the figure legends. Fluorescence values that did not exceed the emission signals from cell-free control samples were set to 1. Specific fluorescence was calculated by taking the quotient of the fluorescence signal per biomass value at a given time point. Specific fluorescence values lower than in the cell-free control samples were set to 0.01. All data presented in the same graphs were obtained in the same BioLector growth experiment using identical gains.

Cell flow cytometer analysis
Reporter gene expression was analyzed by measuring mNG fluorescence on the single cell level with G. oxydans 621H either carrying the plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 , pBBR1MCS-2-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 , or pBBR1MCS-5-P lacI -lacI-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 using a FACSAria™ Fusion cell sorter (BD Biosciences) run with 70 psi sheath pressure and equipped with a 70 µm nozzle. Data acquisition and analysis of the flow cytometer was controlled by the FACSDiva 8.0.3 software (BD Biosciences). Using a 488-nm solid blue laser beam, the forward scatter (FSC) and side scatter (SSC) were employed for cell analysis. Particles/events with FSC-H and SSC-H signals below a threshold of 200 a.u. and 300 a.u. were excluded from the analysis. Detection of emitted mNG fluorescence from the SSC signal was performed by combining a 502-nm long-pass and 530/30-nm band-pass filter. The entire cell population was analyzed in a three-step gating strategy. Initially, the assessed cell population was gated in a FSC-H vs. SSC-H plot, to exclude signals originating from electronic noise and cell debris. From the resulting population, the FSC-H signal was plotted against the FSC-W signal. Subsequently, the obtained population was gated in a SSC-H vs. SSC-W plot, to ensure singlet discrimination. The gated singlet population was used for fluorescence acquisition in all experiments (fluorescence intensity vs. cell count). For all samples, 100,000 events were recorded with an event rate below 10,000 events/s. FlowJo 10.7.2 for Windows (FlowJo, LLC) was used for data analysis and visualization of all gated events (n = 100,000).

Fluorescence microscopy
For fluorescence microscopy, cells were placed on agarose-coated microscope slides and covered by a coverslip. Images were taken on a Zeiss AxioImager M2 imaging microscope that was equipped with a Plan-Apochromat 100 × /1.40-numerical aperture phase-contrast oil-immersion objective and an AxioCam MRm camera. Fluorescence was measured using the 46 HE filter set (λ ex 500/20 nm / λ em 535/30 nm). For all images, identical exposure times were applied. Digital images were acquired and analyzed with AxioVision Rel. 4.8 software (Zeiss).

Total DNA extraction, library preparation, Illumina sequencing, and data analysis
Total DNA was purified from a culture aliquot using a Nucle-oSpin Microbial DNA Mini kit (MACHEREY-NAGEL). DNA concentrations were measured using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). Illumina sequencing libraries of tetR-P tet and lacI-P lacUV5 samples were prepared from 1 µg of isolated DNA using the NEBNext Ultra™ II DNA Library Prep Kit for Illumina according to the manufacturers' instructions (NEB). The libraries were evaluated by qPCR using the KAPA library quantification kit (Peqlab) and then normalized for sample pooling. Pairedend sequencing with a read length of 2 × 150 bases was performed in-house on an Illumina MiSeq system. The demultiplexed sequencing output (base calls) was obtained as fastq files and used for trimming and quality filtering, mapping, and coverage calculation using the CLC Genomics Workbench software (Qiagen). For the mappings, the improved genome sequence from G. oxydans 621H and the tetR-P tet or lacI-P lacUV5 plasmid sequence was used (Kranz et al. 2017).

Computational methods
Homology modeling of TetR, LacI, and AraC structures was performed by YASARA Structure version 19.12.14 (Krieger et al. 2002) using the default settings (PSI-BLAST iterations: 6, E value cutoff: 0.5, templates: 5), and with oligomerization state adjusted to 2 for TetR and AraC, and to 4 for LacI (Altschul et al. 1997). A position-specific scoring matrix (PSSM) was used to score the obtained template structures (Jones 1999;Qiu and Elber 2006). The obtained hybrid models were further evaluated for protein geometry by VERIFY3D and ProSA (Eisenberg et al. 1997;Sippl 1993). Operator models of tetO and araI 1 were generated with Avogadro as B-shaped DNA (Hanwell et al. 2012). The initial coordinates for lacO were taken from the X-ray structure (PDB ID 1EFA, resolution 2.6 Å) (Bell and Lewis 2000).
Surface residues were determined with PyMol script (default cutoff of 2.5 Å 2 ). The protonation states of titratable residues were assigned on the basis of pK a values obtained from the PROPKA 3.1 program for the pH values 7, 6, 5, and 4 (Olsson et al. 2011).
Modeling protein-protein and protein-DNA complexes was performed by using the HADDOCK Webserver (van Zundert et al. 2016). To understand the DNA binding behavior, protein-DNA docking simulations of the transcription factors and their respective operator sequence were done. In docking simulations, a dimeric model for TetR and LacI and a monomeric model for AraC were used. The charge states of proteins were adjusted to the respective pH based on the previous pK a calculations and were adjusted to the respective pH either directly within the PDB file according to the HADDOCK specifications (Asp, Glu), or within the HADDOCK interface (His). Oligomerization of proteins was computed by performing protein-protein docking with the transcription factor subunits, i.e., monomer-monomer docking for TetR and AraC, and dimer-dimer docking for LacI. Due to the fact that the LacI homology model was missing the second tetramerization helix, the dimer structure taken from the crystal structure (PDB ID 3EDC, resolution 2.10 Å) was used for the LacI tetramerization modeling (Stenberg and Vihinen 2009).

A pBBR1MCS-5-based TetR-P tet system was very tight and highly inducible and tunable
The tetR-P tet region including its native RBS from transposon Tn10 was used to construct a pBBR1MCS-5-based reporter plasmid using the reporter gene mNeonGreen (mNG). The tetR gene under the control of its native promoter P tetR overlapping with the divergently oriented TetR-dependent promoter P tet followed by mNG were integrated into the MCS of pBBR1MCS-5-T gdhM -MCS-T 0028 as described in Material and Methods. DNA sequences for transcription terminators were placed adjacent to the tetR-P tet -mNG insert to create transcriptional barriers between the genetic elements on the insert and on the plasmid backbone.
The leakiness and inducibility of P tet were tested in G. oxydans 621H harboring the plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 by omitting and adding anhydrotetracycline (ATc). A pre-culture was split and used to inoculate shake flasks for growth in d-mannitol medium without and with 200 ng mL −1 ATc. The highest mNG fluorescence signals were measured in induced cultures after 10 h of growth at the end of the exponential growth phase, followed by a slight decrease in the stationary phase (Fig. 1a). In the non-induced cultures, the mNG signals barely surpassed background signals of cell-free control samples, suggesting very tight repression of P tet in the absence of inducer. Based on the absolute and the specific mNG fluorescence, the maximal induction ratios were calculated to be 2284 ± 263-fold and 2661 ± 180-fold, respectively. At the end of the cultivation (24 h), cells of an induced culture were harvested and total DNA was purified for Illumina sequencing. In the read data analysis, 99.5% of the reads mapped to the updated reference sequences of the G. oxydans 621H genome, the 5 endogenous plasmids, and the mNG expression plasmid with tetR-P tet (Kranz et al. 2017). Thus, the sequencing results excluded undesired contaminations and verified that the P tet -derived mNG expression was highly induced in G. oxydans 621H carrying the plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 . In microscale BioLector cultivations a similar induction profile of P tet was observed as in shake flasks. The fluorescence in ATc-supplemented cultures peaked approximately after 8 h also followed by a decreased fluorescence level in the stationary phase (Fig. 1b). Again, the basal expression under non-induced conditions was barely detectable and the maximal induction ratio based on the specific fluorescence was calculated to be 3674 ± 193-fold. The drop in mNG fluorescence signals observed in shake flasks and in BioLector cultivations in the stationary phase was caused by the decreased pH of the medium (pH 4.6) and could be largely recovered at pH 6 as outlined below and described previously (Fricke et al. 2020).
To verify that TetR is responsible for the repression of P tet also in G. oxydans, we constructed the plasmid pBBR1MCS-5-T gdhM -P tet -mNG-T BBa_B1002 -T 0028 lacking the tetR gene. In the absence of the plasmid-encoded regulator TetR, in both conditions with ATc supplement and without, the mNG expression from P tet was even higher than in the induced control cultures containing the tetR gene on the plasmid (Fig. S1). This indicated that in G. oxydans P tet is also fully repressed by TetR like in E. coli and that nonblocked P tet is highly active in G. oxydans. Interestingly, even without the tetR gene the addition of ATc resulted in a higher maximal specific fluorescence (28.8 ± 1.3 a.u.) than without ATc (23.5 ± 1.9 a.u). This suggested that in G. oxydans the P tet activity could additionally be affected by an endogenous factor.
We then tested the tunability of the TetR-P tet system in G. oxydans by varying the ATc concentration from 10 to 400 ng mL −1 . Gradual increase of the inducer concentration led to a gradual increase in mNG fluorescence illustrating the high dynamic range of P tet -derived gene expression in G. oxydans with the pBBR1MCS-5 backbone (Fig. 1c). Comparing the peaking fluorescence signals, the system appeared to be almost fully induced with 150 to 200 ng mL −1 ATc. According to flow cytometry analysis (FACS) of samples taken 7 h after induction (Fig. 1a), in both ATc-induced and non-supplemented cultures high population homogeneity was observed (Fig. 1d). Ninety-five percent and 94% of the cells of a sample were found to exhibit either very low (~ 400 a.u., non-induced) or very high (~ 80,000 a.u., ATc-induced) mNG fluorescence signals. To visualize this high homogeneity by microscopy, images of induced and non-induced G. oxydans cells harboring the plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 were taken (Fig. S2). In accordance with the data obtained by flow cytometry, the microscopic images confirmed the strong inducibility of pBBR1MCS-5-based TetR-P tet and the highly homogenous induction response in G. oxydans.
During growth, the G. oxydans d-mannitol medium initially set to pH 6 is acidified to pH 4.7 which causes a loss of intracellular mNG fluorescence suggesting a decreased cytoplasmic pH, at least in the stationary phase (Fricke et al. 2020). We wanted to test experimentally whether an already initially lower medium pH could result in leakiness of P tet in G. oxydans already during growth as observed in Komagataeibacter (Florea et al. 2016). Therefore, G. oxydans carrying the plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 was grown in d-mannitol medium initially adjusted to pH 6, 5, 4 and 3 both in the absence and presence of ATc. After 23 h cells were centrifuged and resuspended in fresh d-mannitol-free medium set to pH 6 to check for potential pH-dependent recovery of Fig. 1 Performance of the TetR-P tet system in G. oxydans 621H. a Growth (OD 600 ) and specific mNG fluorescence in G. oxydans 621H carrying plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 in ATc-induced (200 ng mL −1 ) and non-induced condition in shake flasks. The mNG fluorescence was measured in a Tecan reader (gain 60). The specific fluorescence was calculated from absolute fluorescence per OD 600 . Data represent mean values and standard deviation from three biological replicates with three technical replicates each. b Growth according to backscatter (gain 15) and specific mNG fluorescence (with gain 50) of G. oxydans 621H carrying plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 in ATc-induced (200 ng mL −1 ) and non-induced condition in microscale BioLector cultivations. Data represent mean values and standard deviation from four biological replicates with three technical replicates each. c Graded ATc-dependent mNG expression in G. oxydans 621H carrying plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 in microscale BioLector cultivations. Reporter gene expression measured as fluorescence (gain 50) was induced with increasing concentrations of ATc from 10 to 400 ng mL −1 as indicated. d FACS analysis of G. oxydans 621H carrying plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 or empty vector pBBR1MCS-5 (MCS-5) as a control. Cells were grown in shake flasks with d-mannitol medium without and with 200 ng mL −1 ATc. FACS analysis was performed 7 h after inoculation/induction. Total counts per sample represent 100.000 events mNG fluorescence above the respective levels monitored before that would indicate a leakiness of P tet together with a loss of mNG fluorescence during growth. In all noninduced cultures, very low maximal fluorescence signals (0.6 ± 0.1 to 1.3 ± 0.2 a.u.) were measured independent of the initial medium pH (Fig. S3a-b). Cells grown in pH 3 exhibited the lowest signals and a different growth according to backscatter compared to all other cultures. No sufficient increase of mNG fluorescence was observed in any non-induced condition in the exponential growth phase to suggest a leakiness of P tet in G. oxydans. Moreover, after transfer (23 h) of the cells into fresh d-mannitol-free medium adjusted to pH 6 no recovery of mNG fluorescence above the levels before could be observed. Thus, since the strong induction of P tet by ATc in the cells grown in each medium of this pH series indicated that the mNG protein could always be produced during the exponential phase of growth and exhibited strong fluorescence property ( Fig. S3c-d), the results together indicated that expression from P tet was really tightly repressed in G. oxydans in all pH conditions, even in medium initially set to pH 3.

Terminators strongly affected mNG expression strength but not repression of P tet
Since we did not observe and could not show leakiness of the TetR-P tet system in growing G. oxydans cells per se or in dependence of the medium pH, we wanted to analyze the influence of terminator sequences on the functionality and leakiness of the TetR-P tet system. Therefore, we constructed plasmids lacking terminators downstream from tetR or mNG and both and compared these constructs in regard to P tet -derived mNG expression in G. oxydans (Fig. 2). The terminators downstream from mNG had a major influence on the resulting mNG fluorescence level. With plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG not having T BBa_B1002 and T 0028 downstream from mNG, the mNG fluorescence signals were reduced by half in ATc-induced G. oxydans cells. Compared to the reference plasmid including all terminators, the maximum in specific fluorescence dropped from 19.4 ± 2.9 a.u. to 10.5 ± 0.2 a.u. (Fig. 2e). The specific fluorescence signals of G. oxydans harboring the plasmid pBBR1MCS-5-tetR-P tet -mNG-T BBa_B1002 -T 0028 lacking T gdhM downstream from tetR were significantly (p = 0.0383) higher (22.3 ± 1.1 a.u.) compared to the reference (19.4 ± 2.9 a.u.) suggesting a little positive effect on P tet -derived induction and expression strength when T gdhM downstream from tetR was absent. The construct pBBR1MCS-5-tetR-P tet -mNG lacking all terminators performed similar (9.5 ± 0.7 a.u.) as pBBR1MCS-5-T gdhM -tetR-P tet -mNG lacking only the terminators downstream from mNG (10.5 ± 0.2 a.u.). Hence, in G. oxydans and in case of mNG, termination of the target gene transcription close to the 3' end might be important to achieve higher expression levels in G. oxydans. None of the terminators appeared to affect the repression of P tet in the absence of ATc and a P tet leakiness was not observed.

Comparison of the pBBR1MCS-2 / -5 backbones and insertion of AGG AGA as RBS
The broad-host-range vectors pBBR1MCS-5 and pBBR1MCS-2 differ in the antibiotics resistance they convey for clone selection. pBBR1MCS-5 gmR encodes for a gentamicin-3-acetyltransferase enabling growth on gentamicin and pBBR1MCS-2 neoR/kanR encodes for a neomycin/kanamycin phosphotransferase enabling growth on kanamycin (Kovach et al. 1995). Furthermore, gmR and neoR/kanR are located on different DNA strands which in the case of neoR/kanR could result in transcripts partially overlapping with and antisense to the mRNA of the target gene present in the cloned insert. To check whether the plasmid backbone and antibiotics resistance affect the inducibility of P tet in G. oxydans we compared the performance of the TetR-P tet system with pBBR1MCS-5 and pBBR1MCS-2.
In G. oxydans the pBBR1MCS-2-based TetR-P tet system performed much worse compared to the pBBR1MCS-5-based system despite the similarities of the two plasmids. With pBBR1MCS-2, the specific mNG fluorescence with induced P tet was almost halved and reached 19.2 ± 9.7 a.u. ~ 10 h after induction at the end of the exponential phase of growth, while with pBBR1MCS-5 the maximal fluorescence peaked at 36.7 ± 2.2 a.u. after 8 h (Fig. 3a). Nevertheless, the maximal induction ratios were high with both pBBR1MCS-2 and pBBR1MCS-5 and were calculated to be 1915 ± 757 and 3674 ± 193, respectively. The basal expression under non-induced conditions was not affected by the plasmid backbone. Notably, a much higher standard deviation was obtained with pBBR1MCS-2. FACS analysis with gating of cells in regard to their volume and complexity by forward and side scattering (FSC and SSC) of light revealed that a major part of the G. oxydans cells with the pBBR1MCS-2 derivative and kanamycin exhibited a very different non-typical elongated cell morphology that was not observed with the pBBR1MCS-5 derivative and gentamicin (Fig. 3b). Approximately only 42% of the population with the pBBR1MCS-2 derivative passed the FACS gate where almost 100% of G. oxydans cells without plasmid or with the pBBR1MCS-5 derivative passed. This high heterogeneity of G. oxydans carrying the pBBR1MCS-2 derivative was not affected by the inducer ATc. The occurrence of a portion of elongated cells with pBBR1MCS-2 backbone and kanamycin was also demonstrated by fluorescence microscopy (Fig. S4). Several G. oxydans cells carrying pBBR1MCS-2 or pBBR1MCS-2-tetR-P tet -mNG-T BBa_B1002 -T 0028 appeared to be 10-15 µm and > 40 µm long, while the 621H reference cells typically were 2-3 µm. Apparently, the altered Fig. 2 Variants of pBBR1MCS-5-based expression plasmids with tetR-P tet and reporter gene expression in G. oxydans 621H. a Map of plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 carrying the fluorescence reporter gene mNeonGreen (mNG) expressed from P tet with the adjacent tetR gene and terminators T gdhM , T BBa_B1002 and T 0028 . b Variants of the plasmid insert with reporter gene mNG to test TetR-P tet -dependent expression in the presence and absence of terminators downstream from tetR and mNG, with RBS AGG AGA (Hentschel et al. 2013) inserted in the 3' region of P tet upstream from mNG, and without tetR. c DNA sequence details with P tet region and TetR binding sites (tetO1 and tetO2) and termi-nator sequences adjacent to tetR and mNG. d + e) Growth according to backscatter and specific mNG fluorescence in G. oxydans carrying either plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 (1), or the plasmid lacking T gdhM (2) or T BBa_B1002 -T 0028 (3) or all terminators (4) under non-induced (d) and ATc-induced (e) condition in microscale BioLector cultivations. For induction 200 ng mL −1 ATc was present in the d-mannitol medium. Data represent mean values and standard deviation from two biological replicates with three technical replicates each. T gdhM : terminator sequence of gdhM (GOX0265); T 0028 : terminator sequence of GOX0028. BioLector settings: backscatter gain 20, fluorescence gain 50 cell morphology had no direct effect on the inducibility of the TetR-P tet system in G. oxydans as elongated cells also exhibited strong mNG fluorescence.
To test improved translation and thus resulting reporter protein levels in G. oxydans, we also constructed a plasmid with the RBS AGG AGA that we had also used in the araC-P araBAD system and inserted it upstream from mNG and downstream from tetO 2 overlapping with the native RBS of the P tet region (Fig. 2c). Thus, in this construct, RBS Ptet and RBS AGG AGA were consecutively upstream from mNG. The Fig. 3 Comparison of pBBR1MCS-5 and pBBR1MCS-2 with tetR-P tet . a Growth according to backscatter (gain 15) and specific mNeon-Green (mNG) fluorescence (gain 50) in G. oxydans 621H carrying either plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 or pBBR1MCS-2-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 in microscale BioLector cultivations. For induction, always 200 ng mL −1 ATc was present in the d-mannitol medium. Data represent mean values and standard deviation from at least three biological replicates with three technical replicates each. b Cell morphology according to FACS analysis of G. oxydans 621H type strain (wt) without plasmid and with either the pBBR1MCS-5-or pBBR1MCS-2-based tetR-P tet system, all without the inducer ATc Fig. 4 Influence of the ribosome binding site AGG AGA on mNG reporter expression with the TetR-P tet system. Growth according to backscatter and specific mNeonGreen (mNG) fluorescence in G. oxydans 621H carrying plasmid pBBR1MCS-5-T gdhM -tetR-P tet -RBS-mNG-T BBa_B1002 -T 0028 with the RBS change (a) and with plasmid pBBR1MCS-5-T gdhM -tetR-P tet -mNG-T BBa_B1002 -T 0028 as the control (b) in microscale BioLector cultivations. Cells were grown in d-mannitol medium without or with 200 ng mL −1 ATc for induction. Data represent mean values and standard deviation from two biological replicates with three technical replicates each. BioLector settings: backscatter gain 20, fluorescence gain 40 impact on reporter expression and inducibility was measured in BioLector cultivations. Grown in d-mannitol medium and induced with 200 ng mL −1 ATc, the fluorescence signals of induced cells were further increased and reached > 4000fold induction, yet at such a high level that the detector gain in the BioLector had to be set from 50 down to 40 to avoid signal saturation. For both constructs with gain 40 setting, the insertion of RBS AGG AGA doubled up the specific fluorescence in induced cells from 8.6 ± 0.4 a.u. for the reference to 20.4 ± 0.5 a.u. (Fig. 4). The basal expression in the absence of ATc was again extremely low, and the calculated maximal induction ratio based on the specific fluorescence with gain 40 was increased from 855 ± 7 for the reference to 2042 ± 1 for the plasmid with RBS AGG AGA .

pBBR1MCS-5-based LacI-P lacUV5 was leaking and therefore tunable only up to 40-fold
For the lacI-P lac system, the insert containing lacI under control of its native promoter P lacI , promoter P lacUV5 and lacZα was derived from E. coli BL21(DE3). The promotor P lacUV5 differs from the native E. coli P lac promoter by three point mutations leading to enhanced activity and reduced cyclic adenosine monophosphate (cAMP)-dependency for induction in E. coli (Hirschel et al. 1980). Downstream from the P lacUV5 -controlled reporter gene mNG, the terminator T BBa_B1002 was placed and RBS AGG AGA upstream from mNG (Fig. 5a, b). The lacI-P lac system includes three different operator sites which typically are bound by LacI in the absence of an inducer molecule to repress P lac -derived expression. Of these operator sites, O 1 is located in close proximity to the -10 region of P lac , while O 3 and O 2 overlap with lacI and lacZα, respectively. In E. coli all three operator sites are bound by tetrameric LacI and are required for maximal repression (Oehler et al. 1990). Therefore, in order to enable full repression by LacI, the α-unit region of lacZ with O 2 was included in the insert.
We examined the inducibility of P lacUV5 by IPTG in G. oxydans 621H harboring the plasmid pBBR1MCS-5-P lacI -lacI-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 . Cells were grown in shake flasks using d-mannitol medium supplemented with and without 1 mM IPTG. After 10 h at the end of the exponential growth phase, the highest fluorescence signal was measured. Due to the basal expression in the absence of IPTG, the maximal induction ratios by the absolute and by the specific fluorescence were calculated to be 18 ± 4 and 18 ± 5, respectively (Fig. 5c). When testing the inducibility of P lacUV5 in microscale BioLector cultivations, a similar trend was observed. Under induced conditions, the fluorescence peaked approximately 6 h after induction and subsequently slightly decreased in the stationary phase while in non-induced cultures a basal fluorescence was measured (Fig. 5d). Here, with 1 mM IPTG an average induction ratio of 41 ± 16 was calculated based on the specific fluorescence of the replicates in the BioLector. The P lacUV5 -derived mNG expression exhibited a tunability by IPTG concentrations ranging from 0.1 to 10 mM in G. oxydans (Fig. 5e). The mNG expression from P lacUV5 was gradually increased up to 3 mM IPTG. Above 3 mM IPTG, the mNG expression hardly increased further and thus appeared to be saturated and best-calculated induction ratios were again in the range of 40-fold. Illumina sequencing data obtained with total DNA purified from induced cells showed that 99.5% of the reads mapped to the updated reference sequences of the G. oxydans 621H genome, the 5 endogenous plasmids, and the mNG expression plasmid with lacI (Kranz et al. 2017). Thus, the sequencing results verified that the P lacUV5 -derived mNG expression was induced in G. oxydans carrying the intended plasmid pBBR1MCS-5-P lacI -lacI-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 .
To analyze the induction of P lacUV5 in G. oxydans on the single cell level with regard to the homogeneity of mNG expression, flow cytometer analysis was applied (Fig. 5f). Within the chosen gate, 7 h after induction 95% of all induced cells exhibited high fluorescence (~ 11,000 a.u.) while under non-induced conditions in 96% of all cells, the fluorescence was at the somewhat leaky basal level (~ 1500 a.u.). This indicated that in the exponential growth phase, expression from induced P lacUV5 was very homogenous in G. oxydans. This was also supported by fluorescence microscopy (Fig. S5).
Similarly, as for TetR and P tet , we tested experimentally if initially lower medium pH values could affect the leakiness of P lacUV5 . Therefore, G. oxydans carrying the plasmid pBBR1MCS-5-P lacI -lacI-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 was grown in d-mannitol medium initially adjusted to pH 6, 5, 4, and 3 in the absence and presence of IPTG. After 23 h, cells were centrifuged and resuspended in fresh d-mannitolfree medium of pH 6 to check for potential pH-dependent recovery of mNG fluorescence above the respective levels monitored before that would indicate a higher leakiness of P lacUV5 together with a previous loss of mNG fluorescence already during growth (Fig. S3e-h). In all non-induced cultures, overall basal fluorescence signals up to 10 a.u. were measured with decreasing maxima in the exponential phase when the initial medium pH decreased. Again, cells grown in pH 3 exhibited the lowest signals and a different growth according to backscatter compared to all other cultures. Compared to the pH 6 condition, no sufficient increase of mNG fluorescence was observed in any non-induced pH condition (6 h) in the exponential growth phase to suggest a higher leakiness of P lacUV5 . Additionally, after the transfer (23 h) of the non-induced cells into fresh d-mannitol-free medium adjusted to pH 6 no recovery of mNG fluorescence above the levels with pH 6 could be observed in any condition. Interestingly, only for the non-induced pH 4 and pH 5 conditions, recovery of mNG fluorescence in pH 6 (24 h) above the respective maximal levels observed before in the exponential phase (6 h) were observed. This suggested that in the pH 4 and pH 5 conditions G. oxydans was not able to maintain its typical intracellular pH during growth and mNG fluorescence partially became inactive already in an early phase of growth (6 h).

Strength of P lacUV5 was P lacI -lacI-dependent and leaking was independent of read-through
Unlike most operons, in the E. coli lac operon, the regulatory gene lacI is located immediately upstream from the target operon and is transcribed in the same direction. The repressor LacI forms tetramers binding in the absence of an inducer to up to three operator sites, thereby preventing transcription from the lac promoter directly downstream from lacI. Nevertheless, despite LacI binding and the presence of a lacI terminator region coinciding with the LacI operator site O 1 directly downstream from the − 10 region, readthrough is possible varying from 10 up to 80% (Horowitz and Platt 1982;Oehler et al. 1990). To check the possibility of read-through from P lacI causing the leakiness observed in G. oxydans and interference by endogenous proteins on the P lacUV5 inducibility, we wanted to check and compare the pBBR1MCS-5-based reporter expression in the absence of P lacI -lacI, as well as with P lacI -lacI in opposite orientation toward P lacUV5 .
Firstly, we constructed the plasmid pBBR1MCS-5-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 lacking P lacI and most of the lacI gene, while keeping a short 3' region of lacI to maintain all three operator sites of P lacUV5 including O 3 partly overlapping with the end of the lacI gene. The analysis of the IPTG-dependent inducibility of P lacUV5 in G. oxydans in the absence of P lacI -lacI revealed no difference in fluorescence signals supplemented with IPTG or not (Fig. S6). This indicated that the inducibility observed in G. oxydans solely depended on derepression by LacI. Interestingly, compared to the original system with P lacI -lacI, the maximal reporter expression from P lacUV5 was strongly reduced from 8.4 ± 0.4 to 2.3 ± 0.2 according to the specific fluorescence (6-7 h) when P lacI -lacI was lacking. This suggested that a significant read-through from P lacI appeared to contribute to the expression of the gene downstream from P lacUV5 , thus increasing the apparent P lacUV5 strength in G. oxydans almost fourfold when induced.
Secondly, we constructed plasmid pBBR1MCS-5-lacI-P lacI -P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 with P lacI -lacI in opposite direction to exclude potential read-through toward P lacUV5 -mNG. Despite the opposite directions of P lacI and P lacUV5 , an even somewhat higher leakiness of P lacUV5 was observed when not induced (Fig. S7). Therefore, with 1 mM IPTG the maximal induction fold-change (6.4 h) calculated was only 15.4 ± 0.9 based on the specific fluorescence, while the original P lacI -lacI-P lacUV5 construct reached a maximal induction fold-change (6.7 h) of 39.7 ± 13.9. Surprisingly, the apparent induced P lacUV5 strength was restored according to the reporter fluorescence and reached the same levels as when P lacI -lacI were in the same direction as P lacUV5 . Together, the results indicated that in G. oxydans the promoter strength of plasmid-based P lacUV5 appeared to depend on sequences further upstream and that the P lacUV5 leakiness was independent of potential read-through from P lacI .

Modeling LacI and TetR binding to DNA at acidic pH values
Our results suggested that the leakiness of P lacUV5 in G. oxydans was independent of potential read-through from P lacI and thus should result from transcription initiation from P lacUV5 and insufficient repression by LacI. Since the cytoplasmic pH of G. oxydans may readily be acidified to some extent already during growth when medium pH decreases, we modeled LacI-and TetR-DNA structures at different pH values using YASARA to predict if and how a potentially lower cytoplasmic hydrogen ion activity might affect the binding of LacI and TetR to DNA (Krieger et al. 2002). As a control, the AraC structure was also modeled since an l-arabinose-inducible AraC-P araBAD system was shown to be fully functional in G. oxydans with plasmid pBBR1MSC-5, yet was very leaky in Gluconacetobacter and Komagataeibacter using another expression plasmid (Fricke et al. 2020;Teh et al. 2019). Protein-DNA bindings were simulated in two different steps for pH 7, 6, 5, and 4 (see supplementary Text S1 and S2).
Analysis of the HADDOCK simulations in different pH conditions showed that the HADDOCK scores of all five best-scoring clusters obtained in the TetR-DNA docking simulation for pH 7, 6, 5, and 4 lie within the standard Fig. 5 pBBR1MCS-5-based expression plasmid with lacI-P lacUV5 and reporter gene expression in G. oxydans 621H. a Map of the pBBR1MCS-5-based lacI-P lacUV5 plasmid. b Sequence information details of the lacI-P lacUV5 plasmid. The promoter P lacUV5 comprises the LacI operator sites O 3 , O 1 , and O 2 . The O 3 sites overlaps with the 3' end of lacI. The RBS AGG AGA was inserted upstream from mNG. The iGEM terminator sequence of BBa_B1002 was located downstream from mNG followed by terminator T 0028 from GOX0028. c Shake flasks cultivations of G. oxydans 621H carrying plasmid pBBR1MCS-5-P lacI -lacI-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 in IPTG-induced (1 mM) and non-induced condition. The mNG fluorescence was measured in a Tecan reader (gain 60. The specific fluorescence was calculated from absolute fluorescence per OD 600 . d Microscale BioLector cultivations of G. oxydans 621H carrying plasmid pBBR1MCS-5-P lacI -lacI-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 in IPTG-induced (1 mM) and non-induced condition. All data represent mean values and standard deviation from three biological replicates with three technical replicates each. Backscatter gain 15, fluorescence gain 60. e Microscale BioLector cultivations of G. oxydans 621H carrying plasmid pBBR1MCS-5-P lacI -lacI-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 with increasing concentrations of IPTG as indicated.
Fluorescence gain 60. f FACS analysis of G. oxydans 621H carrying plasmid pBBR1MCS-5-P lacI -lacI-P lacUV5 -RBS-mNG-T BBa_B1002 -T 0028 . Cells were grown in shake flasks with d-mannitol medium and 1 mM IPTG. FACS analysis was performed 7 h after inoculation and induction. Total counts per sample represent 100.000 events. As a control G. oxydans 621H carrying the empty vector pBBR1MCS-5 was used (MCS-5) ◂ deviation of each other. To make a prediction of the DNA binding behavior in dependence of the pH value, similar docking positions were identified for each pH value and the cluster scores were compared. It can be observed that with decreasing pH values the scores decrease. The smaller the score, i.e., the more negative, the better is the predicted binding to DNA, suggesting an increased binding of TetR to DNA (Fig. 6). Similar results were obtained for AraC. Thus, when comparing the scores of clusters with corresponding docking positions, the values generally decreased with decreasing pH values, suggesting a generally increased binding to DNA for both TetR and AraC (Fig. 6). On the contrary, for LacI unique clusters with correlating docking poses identified for pH 7, 6, 5, and 4 exhibited increasing HADDOCK scores for decreasing pH values, suggesting a decreased DNA binding of LacI when pH decreases ( Fig. 6 and Supplementary Text S1 and S2).

Discussion
In this study, we found with a pBBR1MCS-5-based TetR-P tet system that inducible target gene expression based solely on de-repression of the heterologous target promoter can also perform extremely well in G. oxydans. With the pBBR1MCS-5-based plasmid constructed here, the anhydrotetracycline (ATc)-inducible promoter P tet derived from the E. coli transposon Tn10 exhibited excellently tunable expression performance in an inducer concentrationdependent manner with maximal induction ratios up to more than 3500-fold. This was due to extremely low basal reporter expression in the absence of an inducer, thus well repressed P tet , and P tet being very strong in G. oxydans.
In contrast, expression from P lacUV5 was always leaky when not induced and this appeared to be independent of potential read-through from P lacI which was located with lacI immediately upstream from P lacUV5 and, unlike most regulator genes and target operons, coupled very close and transcribed in the same direction. Therefore, the transcription termination by the native lacI terminator is expected to be very effective in G. oxydans and the P lacUV5 leakiness is rather related to the early events after transcription initiation from P lacUV5 . In E. coli, the LacI repressor is not preventing per se the binding of RNA polymerase (RNAP), but is blocking the progress of RNAP which has bound to the DNA and has begun transcribing (Reznikoff et al. 1969). Transcription through roadblocks has been shown to depend on the cooperation of the leading and trailing RNAP molecules and thus depends on how many RNAP molecules are allowed to initiate from the same promoter upstream from the roadblock (Epshtein et al. 2003;Hao et al. 2014). In the case of P lac or P lacUV5 , the distance from the transcriptional start (TSS) to the LacI binding site O 2 is rather short and site O 1 is even overlapping with the TSS; thus, only one RNAP molecule is expected to initiate upstream from the roadblock. Additionally, the E. coli σ 70 subunit of RNAP mediates a pause at the lac promoter closely downstream from the transcriptional start which is proposed to function in limiting the downstream gene expression (Nickels et al. 2004). If the G. oxydans σ 70 subunit of RNAP also mediates this pause at the lac promoter is unknown. Nevertheless, RNAP cooperation should not be possible and thus not contribute to the leakiness of P lacUV5 in G. oxydans, yet the extent of read-through can also be affected by accessory factors. For example, in E. coli GreA and GreB rescue backtracked (roadblocked) RNAP by cleavage of the RNA, regenerating a new 3' end at the catalytic site, and can aid passage of RNAP through a LacI roadblock in cells (Toulme et al. 2000). Furthermore, in E. coli the transcription-coupled repair protein Mfd binds to the DNA behind RNAP and uses ATP to push backtracked RNAP forward until the 3' end of RNA is back at the catalytic center, yet the forces generated by Mfd may also result in RNAP termination (Park et al. 2002 read-through behind roadblocks in G. oxydans and AAB in general, yet the leakiness of P lac or P lacUV5 is likely rather related to weaker binding of LacI to operator DNA and thus weaker promoter repression in G. oxydans. Our docking simulations predicted that below pH 6 the binding of LacI to DNA is decreased which could cause or contribute to promoter leakiness. Neutralophilic bacteria maintain an intracellular pH between 7 and 8, while in acidophilic bacteria intracellular pH is considered to be maintained between 6 and 7 (Baker-Austin and Dopson 2007; Krulwich et al. 2011). The mannitol LB medium typically used for G. oxydans is initially set to pH 6 and can be acidified during growth in dependence of the carbon and energy sources, for example, to approximately pH 4.5 or 3.3 as well (Fricke et al. 2020). In such acidic pH conditions of the growth medium strongly decreased, intracellular fluorescence reporter activities of mNeonGreen (mNG) were observed in G. oxydans in the stationary phase (Fricke et al. 2020). Studies with isolated mNG revealed a strong decrease in fluorescence at pH 4.4 and below (Steiert et al. 2018). Therefore, the decrease in mNG fluorescence in G. oxydans cells was attributed to an acidified intracellular pH, at least in the stationary phase. The loss in mNG fluorescence in stationary G. oxydans cells can quickly be restored without protein synthesis just by short incubation in pH 6 condition as shown in this study and before (Fricke et al. 2020). Moreover, for the AAB Acetobacter aceti, the intracellular pH was reported to change from 5.8 to 3.9 already during growth when the medium pH acidified from 6.2 to 3.5 (Menzel and Gottschalk 1985). Despite intracellular pH maintenance mechanisms, it is conceivable that depending on the growth conditions in some or many AAB the cytoplasmic pH can decrease already during growth below the range of pH 6 to 7 considered to be typically maintained in acidophilic bacteria. According to our data, the mNG reporter fluorescence in G. oxydans cells with LacI-P lacUV5 grown in non-induced pH 4 and pH 5 conditions suggested that G. oxydans was not able to maintain its typical intracellular pH during growth and the mNG fluorescence became partially inactive already in the middle phase of growth (6 h). To get LacI-dependent systems tight and suitable for use in G. oxydans or AAB in general, the LacI protein may require engineering for increased binding to DNA in acidic pH condition. However, more intracellular pH data of AAB strains in various growth conditions are required. Besides leakiness, in G. oxydans, the strength of fully induced P lacUV5 was much lower compared to that of the fully induced P tet (compare absolute fluorescence Fig. S3d and S3h). It was already reported before that P lac is rather weak in G. oxydans 621H and the P lacUV5 mutant promoter exhibiting three base pair changes including two changes at positions − 9 and − 8 apparently also exhibits a similar low expression strength in G. oxydans (Hirschel et al. 1980;Kallnik et al. 2010). Thus, in G. oxydans the TetR-P tet system will probably always be the choice for applications because of its extremely low basal expression and very high dynamic range of the tunable expression strength from P tet .
Interestingly, in K. rhaeticus iGEM, the TetR-P tet system tested exhibited high leakiness in the absence of the inducer (Florea et al. 2016). In G. oxydans, we could neither observe nor demonstrate relevant leakiness of TetR-P tet , not with the pBBR1MCS-5-based construct variants (± terminators), not with the pBBR1MCS-2 backbone and kanamycin causing abnormal cell morphology, and not in the media of the pH series. Without terminators downstream from mNG, the length of the mNG transcript is assumed to exhibit an extended 3' UTR. This appeared to only decrease transcript stability or translation, since the mNG signals were reduced to half, yet repression of P tet was not affected. For the tetR transcript, it is unknown how stability and translation are affected in G. oxydans when the 3' UTR is longer or shorter, yet altered repression of P tet was also not observed with either of the constructs. A major difference of the two tetR-P tet constructs tested in K. rhaeticus iGEM and in G. oxydans is that we maintained the opposite orientation of tetR-P tetR and P tet as originally present on Tn10, while in K. rhaeticus iGEM the construct tested contained the promoter J23118 or P lacI for expression of tetR followed by a terminator sequence and all directly upstream from the target promoter P tet and in the same direction as P tet (Florea et al. 2016). It was assumed that the terminator is effective, yet it seemed to be unknown to what extend the terminator allowed potential read-through in these constructs. This could possibly cause the high leakiness of P tet in K. rhaeticus iGEM, especially when cooperative RNAP molecules read through the TetR roadblock. On the other hand, our computational simulation also predicted that with decreasing pH value TetR dimerization decreases (Text S1 and Text S2). That could finally affect binding to DNA and thus promoter repression since TetR binds as a dimer (Hillen and Berens 1994). Thus, if the prediction holds true, in G. oxydans TetR dimerization is not sufficiently negatively affected by mild acidic intracellular pH. Komagataeibacter is typically grown in Hestrin − Schramm (HS) medium with glucose or in LGI medium with sucrose, both initially set to pH 6 or pH 4.5 and acidified further during growth (Florea et al. 2016;Hestrin and Schramm 1954). The intracellular pH of A. aceti was reported to change from 5.8 to 3.9 already during growth when the medium pH acidified from 6.2 to 3.5 (Menzel and Gottschalk 1985). The extent of being able to maintain the intracellular pH not only in the stationary phase but also during exponential growth could differ between AAB species. This could possibly be seen in the results with the LuxR-P lux system in Komagataeibacter (Florea et al. 2016). The system exhibited condition-dependent induction only up to fivefold due to high leakiness, yet it also exhibited extremely low leakiness and much better induction performance according to fluorescence microscopic analysis in cells inside cellulose pellicles. Cellulose pellicles generate a micro-environment with the advantage of potentially protecting the cells from harsh conditions which could also include protection from acidic environmental pH. Again, to better understand the pH-dependent binding of regulators to DNA and to improve the expression performance of heterologous regulatable expressions systems so far leaky in AAB and their growth conditions, more intracellular pH data of AAB strains are required for various growth conditions. Additionally, experimental pH-dependent protein-protein and protein-DNA interaction data are needed.
In summary, we could show that in G. oxydans the TetR-P tet system tested was extremely well performing with the pBBR1MCS-5 backbone and exhibited very strong reporter expression when induced, while the LacI-P lacUV5 system was always leaking and thus resulting in much lower induction ratios. Therefore, the pBBR1MCS-5-based TetR-P tet system with the low inducer concentrations required for gradually tuning target gene expression appeared to be highly suitable for applications in G. oxydans and possibly in other AAB.