Evolved sequence features within the intrinsically disordered tail influence FtsZ assembly and bacterial cell division

Megan C. Cohan; Ammon E. Posey; Steven J. Grigsby; Anuradha Mittal; Alex S. Holehouse; Paul J. Buske; Petra A. Levin; Rohit V. Pappu

The CTL variant strains were constructed as described previously (Buske and Levin, 2013) . The bacterial strains and plasmids used in this study are listed in Table S1 of the supplemental material. Synthetic double-stranded oligonucleotides of the CTL variants were ordered from Integrated DNA Technologies, digested using restriction enzymes, and ligated into pPJ19, which contains FtsZ, under the control of the Pspac promoter that is inducible with 0.1 mM IPTG, with restriction sites flanking the CTL. A BamHI site after amino acid 315 and a XmaI site before residue 366 result in the insertion of amino acid pairs GS and PG N-and C-terminal to the CTL, respectively. The plasmid was transformed into PAL 644, a strain of E. coli derived from PAL930. PAL 644 contains the low copy plasmid pBS58 expressing E. coli ftsQAZ, which allows the sub-cloning of B. subtilis FtsZ. FtsZ was amplified in pPJ19, the product of which was restriction digested, purified, and ligated into pDR67. The multiple cloning site in the vector pDR67 contains the 5'-and 3'-ends of the amyE gene on either side, which allows the insertion of FtsZ into the amyE locus by homologous recombination.