## 2 Materials and Methods  
Bacterial strains and plasmids. The strains used in this study were the curdlan producing _agrobacteriumium_ sp. strain ATCC 31749, whose genome has been sequenced [54] and _agrobacteriumium tumefaciens_ C58 [55]. Mutants were generated using a chromosomally inserted suicide plasmid (pK18 mob2) [56]. For transferring plasmids into the _agrobacteriumia_, the _E. coli_ strain S17-1 was used for conjugation. Plasmid construction was performed using the restriction digest sites indicated in Figure 1.  
Upon homologous recombination with the chromosome, the suicide plasmid inserts an additional copy of the homologous fragment carried on the plasmid. For this approach, I used different fragments from the _crdASC_ gene cluster. Firstly, to learn about the expression of this cluster, a copy of the promoter region of _crdA_ (upstream 300 bp) plus the first 51 bp of the coding region of _crdA_ (351 bp total) was fused to the gene for the reporter protein mVenus. This translational fusion construct was inserted into the chromosome using the suicide vector pK18 mob2 and homologous recombination (see Figure 1A, suicide plasmid 1, reporter plasmid). Upon insertion, the CrdA::mVenus fusion provided a readout of _crdA_ expression. Importantly, the insertion created an additional copy of the _crd_ promoter (Pcrd) without interrupting the native _Pcrd-crdASC_ expression and curdlan production. Another fragment of _crdA_ was used in the second and third suicide variants (see Figure 2B,C, suicide plasmids 2 and 3). For suicide plasmid 3, this _crdA_ fragment (489 bp) was preceded by a fragment from the bacterium _R. spharoides_ encoding the _phaP_ promoter (PphaP, 127 bp). Upon homologous recombination with the chromosome, this suicide plasmid variant inserts PphaP upstream of the _crdASC_ gene cluster, meaning that expression of the cluster is now dependent upon PphaP. The DNA sequences of the suicide plasmids used in this study are included in a text file as in Supplementary File S1. For the measurement of the activity of Pcrd and PphaP using the reporter gene for mVenus, the low copy plasmid pPHU231 was used, in which either Pcrd or PphaP was fused to the gene for mVenus, as previously described [52]. In the case of Pcrd::mVenus, the fusion was a translational fusion, and was the identical fragment which was used for the suicide vector pK18 mob-Pcrd::mVenus (see Figure 1A). This fragment included the ribosome-binding site of _crdA_ plus the first 27 codons of _crdA_ (including the translational start). In the case of PphaP::mVenus, the fusion included the native ribosome binding site of _phaP_ plus the first 13 codons of _phaP_ (including the translational start). A comparison of the two ribosome binding sites reveals some similarity: CAGGAGACAAGGCAatg for _phaP_ and GAGGAGTTGATTTCatg for _crdA_ (ribosome binding sites are underlined). Figure 1: Genetic modifications of the _crdASC_ locus. Three versions of the suicide plasmid (**A–C**) with highlighted differences for ensuring genetic modification at the _crdASC_ locus. (**A**), the _crdA_::mVenus reporter construct (suicide plasmid 1, translational fusion) is inserted into the chromosome via homologous recombination (indicated with dashed line) using the PcrdA fragment fused to the gene for mVenus (in yellow). The insertion of this construct provides a read-out of _crdA_ expression without disrupting the _crdASC_ gene cluster. (**B**), suicide plasmid 2 includes a 507 bp fragment from the _crdA_ open reading frame for guiding homologous recombination. The fragment includes the ribosome binding site and the translation start of _crdA_ (total length of _crdA_ = 1458 bp). Insertion of this construct effectively knocks out (KO) transcription of the _crdASC_ gene cluster, since this cluster lacks now lacks a promoter, while the Pcrd controls only a fragment of _crdA_. (**C**), the strong PphaP promoter sequence is fused to the fragment of _crdA_.