Generation of In-frame Deletion Mutants

In-frame deletion mutants were generated using the suicide plasmid pEXG2 as described in Klein et al.^{34 (link)} or its derivate pEXTK. In pEXTK, the sacB gene is replaced by a thymidine kinase gene. If pEXTK based mutator plasmids were used in the mutagenesis procedure, the positive selection to obtain a second crossover was performed by incubating merodiploidic clones for 3 h in 5 ml LB medium containing IPTG (1 mM). Subsequently, bacteria were positively selected by streaking bacteria on LB agar plates containing 200 µg/ml azidothymidine (Acros Organics) and 1 mM IPTG. Bacteria were then tested for loss of gentamicin sensitivity and mutants were verified by PCR as described previously^{34 (link)}. In brief, genomic DNA was isolated using the DNeasy Ultraclean Microbial Kit (Qiagen) and approximately 20 ng DNA was used to perform PCR using Mango Mix (Bioline), primers (Sigma-Aldrich) and water. Two primer pairs were used, namely (i) gene of interest (GOI) seqF and GOI seqR and (ii) GOI seqF and GOI insideR to distinguish between wildtype and mutant. The generated mutants and the primers used in this study are listed in Supplementary Data 1 or 2, respectively.