Isolation and characterization of Kil-resistant <i>zipA</i> alleles.

<p>(A) The <i>zipA_L286Q</i> allele is a spontaneously isolated mutant resistant to loss of viability caused by <i>kil</i> expression from dual plasmids. Shown is an image of an LB plate with 1 mM IPTG and 0.2% arabinose to induce <i>kil</i> from pRR48 and pBAD33 simultaneously, with the original strain (left) and the <i>kil</i>-resistant isolate (right). Targeted sequencing of the <i>zipA</i> locus identified the resistant mutation as <i>zipA_L286Q</i>. (B) Location of L286 and Q290 of ZipA in the co-crystal structure of the <i>E. coli</i> ZipA C-terminal domain (ZipA_C) with an <i>E. coli</i> C-terminal FtsZ peptide (FtsZ_C) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004217#pgen.1004217-Mosyak1" target="_blank">[58]</a>, as generated by Chimera (<a href="http://www.cgl.ucsf.edu/chimera/" target="_blank">http://www.cgl.ucsf.edu/chimera/</a>). For ZipA_C, α-helices are colored red and β-sheets are colored purple. The L286 sidechain is indicated in green and the Q290 sidechain in blue. FtsZ_C is colored cyan. (C) ZipA_WT and ZipA_L286Q expressed from pKG110 are made at levels comparable to native ZipA. Shown is an immunoblot against ZipA (upper panels) from whole cell extracts of indicated strains harvested during mid-log growth in the presence of 0.5 µM sodium salicylate. (D) Replacement of the native <i>zipA</i> gene with <i>zipA_L286Q</i> or <i>zipA_L286R</i> by recombineering confers Kil resistance. Spot dilutions of indicated <i>zipA</i> alleles in a CC4506 background strain are shown under conditions where <i>kil</i> is not expressed (32°C, left) and expressed (42°C, right). (E) Cells harboring <i>zipA_L286Q</i> are resistant to Kil specifically, and not to MinCD or SulA. Representative DIC micrographs of WM1074 cells with the indicated plasmids are shown under inducing conditions (0.2% arabinose for pBAD33; 1 mM IPTG for pDSW210). Scale bar = 5 µm.