Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum

Abstract

The majority of the genes present in bacterial genomes remain poorly characterised with up to one third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to easily transformable species leaving those with low DNA-transfer rates out of reach. Here we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as a sole carbon and energy source.

Design, Analysis, and Implementation of a Novel Biochemical Pathway for Ethylene Glycol Production in Clostridium autoethanogenum

“…First, the genes required for expressing all the pathway enzymes were placed as a synthetic operon, under the control of the P tdcB promoter. 46 This lactose-inducible system allows fine-tuned target expression as previously described. 46 For this step, the 4-step (reactions 3–6) and 6-step (reactions 1–6) versions of the pathway ( Figure 2 ) were compared to confirm that overexpression of the first two reactions already present in C. autoethanogenum would not improve yield as per the computational predictions.…”

“… 46 This lactose-inducible system allows fine-tuned target expression as previously described. 46 For this step, the 4-step (reactions 3–6) and 6-step (reactions 1–6) versions of the pathway ( Figure 2 ) were compared to confirm that overexpression of the first two reactions already present in C. autoethanogenum would not improve yield as per the computational predictions. Unfortunately, these synthetic operons did not allow EG production (data not shown).…”

“…To overcome this obstacle, the genes, aceA , ghrA , and aldA from E. coli , corresponding to reactions 3, 4, and 5, respectively ( Figure 2 ), were expressed as a synthetic operon controlled by the inducible P tcdB promoter, while fucO , coding for the last reaction of the pathway ( Figure 2 ), was placed under the control of the theophylline-inducible riboswitch 47 on a separate plasmid (Figure S1 in the Supporting Information ). In this approach, expression at the P tcdB promoter was controlled by the Clostridiodes difficile sigma R factor TcdR, inserted in C. autoethanogenum genome and controlled by the lactose-inducible promoter P bgaL , itself activated by the transcriptional regulator BgaR to allow a two-level expression control, as described by Woods et al 46 Therefore, induction of genes regulated by P tcdB was achieved by addition of lactose in the medium. Moreover, the riboswitch used for fucO expression prevented gene expression in the absence of theophylline by forming a stem loop structure sequestering the ribosome-binding sequence (RBS); thereby, preventing ribosome binding and gene expression.…”

“…The medium was also supplemented with 500 μg/mL of erythromycin, 25 μg/mL of chloramphenicol, and 50 μg/mL of kanamycin where appropriate. C. autoethanogenum DSM 10061 C24 (henceforth C. autoethanogenum C24) 46 and its derivatives were grown in YTF (10 g/L yeast extract, 16 g/L tryptone, 10 g/L fructose, 0.2 g/L sodium chloride, 1 mL vitamin solution, 1 mL trace element solution, pH 5.8), solidified with 15 g/L of agar and supplemented with 6 μg/mL of clarithromycin and 7.5 μg/mL of thiamphenicol when needed. 5 mM of β-lactose and 5 mM of theophylline were added to induce gene expression when required.…”

“…aceA , ghrA , and aldA were cloned into pMTL83251 with SacI-SpeI, SpeI-HpaI, and HpaI-XbaI, respectively, downstream of the P tdcB promoter. 46 fucO was fused to the riboswitch-P fdx promoter 47 in an NEBuilder HiFi DNA Assembly (New England Biolabs, UK) reaction and cloned into pMTL84151 with NotI and NheI. Cloning steps were performed in E. coli TOP10.…”