Lightrun tube

Bacterial cells from a single colony were suspended in 100 μl TE-buffer and 100 μl lysis buffer (1% Triton X-100, 0.5% Tween 20, 10 mM Tris-HCl with pH 8 and 1 mM EDTA) [32 (link)]. The mixture was incubated at 95°C for 15 minutes and centrifuged at 14 500 rpm for 2 minutes before 100 μl of the supernatant was transferred to a new tube. This material was used as template in the PCR reaction, with AmpliTaq Gold DNA Polymerase with Buffer I (5U/μl; Applied Biosystems). The sar5-specific primers used were identical to those of Mavenyengwa et al; sar5 forward and sar5 reverse [8 (link)]. For three representative strains (CCUG 29784 as the positive control strain, 93–33 as an R3-negative sar5-positive strain, and 93–50 as an R3-positive sar5-positive strain) the PCR products were sequenced using the sar5 forward primer and the Eurofins Genomics sequencing services (LightRun Tube format).

The fructose transporter operon from A. variabilis (Ava_2170-Ava_2173) was amplified from genomic DNA with the Gibson primers 3+4 (sequence in Table 2), adding overlapping fragments to the operon. Vector pRL1049, which is self-replicating in Anabaena sp. (Black and Wolk, 1994 (link)) was digested with EcoRI and BamHI. After that, the fragments were fused using Gibson Assembly (Gibson, 2011 ) and transformed in E. coli TOP10. The construct was verified by sequencing (LightRun Tube, Eurofins Genomics). Transformation in Anabaena sp. was performed by conjugation as previously described (Elhai and Wolk, 1988 (link)). The presence of the replicative plasmid in Anabaena sp. PCC 7120 was verified by a colony PCR using the primers 5+6 (sequence in Table 2). The strain was cultivated in the presence of 5 μg^∗mL^–1 spectinomycin and 5 μg^∗mL^–1 streptomycin.

To analyze the genomic background of the spontaneous A. variabilis-7dSh^R mutant, genomic DNA of the mutant and the wildtype was isolated and the genomic environment of the fructose ABC-transporter operon (Ava_2170-Ava_2173) was investigated (see Supplementary Figure 2). Gene specific primers were designed for each single gene of the operon, but also for the whole operon. A PCR with a Taq polymerase (RedTaq Mastermix, Genaxxon bioscience, Ulm, Germany) was performed to amplify these regions. The products were analyzed on an agarose gel.
Genomic DNA of the Synechocystis sp.-7dSh^R mutant and wildtype was amplified with a Q5-Polymerase (New England Biolabs, Ipswich, MA, United States) using primers 7+8 (sequence in Table 2). The product was analyzed by Sanger sequencing (LightRun Tube, Eurofins).
To obtain high quality genomic DNA of S. elongatus wildtype and S. elongatus-7dSh^R, the extraction was performed with the DNeasy PowerLyzer Microbial Kit (Qiagen). Whole genome sequencing was performed by CD Genomics (“Microbial Whole Genome Sequencing”, New York, NY, United States).