## 2 Materials and Methods
Insertion of suicide plasmid 3 via homologous recombination ensures overexpression of the _crdASC_ gene cluster due to the insertion of the strong PphaP promoter directly upstream of _crdA_. (**D**), alignment of the DNA sequences of the _crd_ and _phaP_ promoters. Highlighted in grey are the \(-35\) and \(-10\) promoter elements. The transcription start of PphaP (+1, bold, underlined) was identified based on RNA sequencing data from _R. sphareoides_[57]. The transcription start of Pcrd was based on [PERSON] et al. [35]. Media and culture conditions. _Agrobacteriumum_ strains were routinely grown at 30 \({}^{\circ}\)C for a minimum of 3 d on aniline blue agar (ABA) [34]. For liquid cultures (shaken at 130 rpm), strains were grown in Luria broth (LB, 10% Oxoid bacto-tryptone, 5% Oxoid yeast extract, 10% NaCl) or _agrobacteriumum_ defined broth (ADB). ADB is medium that is weakly buffered with a phosphate solution, so that initial growth of _agrobacteriumum_ is around pH 7, which then subsequently drops to around pH 5.5 at the onset of curdlan production [34; 50]. ADB consists of 4% (\(w/v\)) glucose (or 4% mannitol), 20 mM KNO\({}_{3}\), 2.8 mM K2 HPO\({}_{4}\), 12.8 mM KH\({}_{2}\)SO\({}_{4}\), 11.5 mM Na\({}_{2}\)SO\({}_{4}\), 1.25 mM MgCl\({}_{2}\), 0.09 mM FeCl\({}_{3}\), 0.05 mM MnCl\({}_{2}\), 1.0 mM citric acid, adjusted to pH 7.0 with NaOH before autoclaving. (Sterile glucose was added to the salts solution after autoclaving.) Figure 2: Curdlan forms a capsule. _Agrobacteriumum_ sp. 31749 cells were grown for 7 days in _agrobacteriumum_ defined broth (ADB) and subjected to microscopic analyses. Cells were stained with aniline blue fluorophore and photographed with bright field ((**A**), bar = 5 \(\upmu\)m) and fluorescence ((**B**), excitation at 330–385 nm, emission at 420–475 nm). (**A**,**B**) photographs are from the same field. (Note that this sample was prepared by firmly pressing the glass slide and the object cover together, resulting in the disruption of some cells from the cell aggregates). Cells grown in ADB from strain 31749 (**C**) and 31749 _crdS_ disruptant (**D**) were stained with uranyl acetate and viewed using electron microscopy (bar = 2 \(\upmu\)m). These strains were also subjected to scanning electron microscopy ((**E**,**F**), bar represents 200 nm). The curdlan capsule appears as a dark cloud in (**C**), and an irregular outer layer in (**E**). The capsule is absent in a _crdS_ mutant (**D**,**F**). MOPS-buffered medium was previously used for growth of _sinorhizobium melitol_ in a defined medium [58] and also supported curdlan production by _agrobacteriumum_. The MOPS medium consists of 50 mM MOPS (adjusted to pH 7 with KOH), 55 mM mannitol, 5 mM Na glutamate (or 20 mM for excess N), 250 mM CaCl\({}_{2}\).2H\({}_{2}\)O, 37 \(\upmu\)M FeCl\({}_{3}\).6H\({}_{2}\)O, 48 \(\upmu\)M H\({}_{3}\)BO\({}_{3}\), 10 \(\upmu\)M MnSO\({}_{4}\).H\({}_{2}\)O, 1 \(\upmu\)M NaMoO\({}_{4}\).2H\({}_{2}\)O, 0.3 \(\upmu\)M CoCl\({}_{2}\).6H\({}_{2}\)O, 2 mM K\({}_{2}\)HPO\({}_{4}\), and 4 \(\upmu\)M biotin. Microscopy. Fluorescence microscopy: An Olympus BX50 microscope with a xenon lamp and on a Zeiss Universal light microscope with ultraviolet light (365 nm from an OSRAM HBO-100 lamp) were used. Images were captured with an RT Monochrome SPOT camera (Diagnostic Instruments Inc., Foxboro, MA, USA). Complexes of curdlan with the (1\(\rightarrow\)3)-\(\beta\)-glucan specific, Aniline Blue Fluorochrome (ABF) (Biosupplies Australia Pty. Ltd., Bundoora, Australia) were detected by mixing one drop of a 7-day culture from ADB with one drop of a 0.01% ABF solution on a glass slide, covering with a coverslip, which was then firmly pressed onto the slide. Fluorescence from the ABF-curdlan complex was observed with excitation at 330-385 nm and emission at 420+ nm. For vital staining: 10 mL of a 4d-ADB culture was incubated with 5 mM 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) [59] for 1 h on a shaker (130 rpm) at room temperature.