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.

The l-rhamnose-dependent regulator RhaS and its target promoters from Escherichia coli expand the genetic toolkit for regulatable gene expression in the acetic acid bacterium Gluconobacter oxydans

“…Furthermore, downstream from the native ribosome binding site (RBS) present in P rhaBAD the RBS 5′-AGGAGA was inserted upstream from mNG. This RBS appeared strong in G. oxydans and was also used in the regulatable AraC-P araBAD and TetR-P tet expression systems (Fricke et al, 2020(Fricke et al, , 2021b. The inducibility of the resulting plasmid pBBR1MCS-5-rhaSR-P rhaSR -P rhaBAD -mNG was tested in G. oxydans 621H with 1% (w/v) l-rhamnose.…”

“…In this study, all plasmids were constructed using the vector pBBR1MCS-5-T gdhM -MCS-T GOX0028 that we created previously for the TetR-P tet system ( Fricke et al, 2021b ). The terminator sequences of GOX0265 (T gdhM ) and GOX0028 (T GOX0028 ) flank the multiple cloning site (MCS) to reduce potential interferences caused by genetic elements on the plasmid backbone.…”

“…DNA concentrations were measured using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). Illumina sequencing and data analysis of the indicated P rhaBAD DNA sample was carried out as described ( Fricke et al, 2021b ). For the read mapping, the improved genome sequence from G. oxydans 621H and the indicated P rhaBAD plasmid sequence were used ( Kranz et al, 2017 ).…”

“…For expression, derivatives of the pBBR1MCS plasmid family obtained from the endogenous plasmid pBBR1 from Bordetella bronchiseptica were the most successful shuttle and expression vectors used (reviewed in Fricke et al, 2021a ). Since pBBR1MCS-2 conferring kanamycine resistance typically results in an abnormal cell morphology of G. oxydans in the presence of kanamycin and potentially also in reduced expression performance, pBBR1MCS-5 and the use of gentamicin is advantageous ( Fricke et al, 2021b ). However, both plasmid backbones recently enabled high functionality of transferred heterologous expression systems for regulatable target gene expression in G. oxydans for the first time.…”

“…Therefore, the typical repression of P araBAD by AraC in the absence of the inducer l -arabinose was not required to ensure non-induced tightness of P araBAD in G. oxydans . Secondly, the TetR-P tet system in its native divergent organization as present in the E. coli transposon Tn 10 exhibited extremely low basal expression in G. oxydans and achieved more than 3,500-fold induction according to reporter assays using the fluorescence protein mNeonGreen ( Fricke et al, 2021b ). In contrast to P araBAD and AraC, P tet highly required the repression by its regulator TetR for tightness of the system, otherwise the expression from P tet was very strong in G. oxydans without TetR.…”