Bacterial morphology is closely related to the environment (1). A change of cell shape may result in reduced cell growth and viability. E.coli has at least three cytoskeletal proteins, MreB, FtsZ, and RodZ; their function is to maintain the organism's rod shape. Although FtsZ and MreB have been extensively studied (2), the functions and molecular mechanisms of RodZ are largely unknown. Cells lacking rodZ are round or oval and grow more slowly than the wild-type (3–5). Furthermore, Bendezu et al. found that, below 30°C, rodZ deletion mutants fail to form colonies on L plates (3). We noticed that, in a culture of rodZ deletion mutant cells (JW2500) in a deletion library (the Keio collection) (5, 6), some cells had formed colonies on L plates below 30°C. We surmised that an additional mutation had suppressed only the cold-sensitive growth of the rodZ mutant, because the cells that had formed colonies were still round. Therefore, the whole genome of a revertant (DS339) was sequenced. We found two mutations aside from the rodZ deletion mutation: a mutation in the ispA gene (ispA-A164T) and one at two base pairs downstream of rrfG, which is one of eight genes encoding 5S ribosomal RNA. However, the rrfG mutation proved not to be a suppressor mutation of cold-sensitive growth of the rodZ mutant (see below). In our experiments, only the ispA-A164T mutation suppressed cold-sensitive growth of the rodZ mutant. This does not exclude the possibility that another mutation of ispA or other mutated genes can suppress cold-sensitive growth of the rodZ mutant. Further isolation of the suppressors is necessary to confirm this.
To test whether cold-sensitive growth of the rodZ mutant was suppressed by the ispA mutation, we constructed the following strains by P1 transduction. All strains are derivatives of BW25113, which is a lineage of E. coli K-12 (6, 7). To restore the wild-type rodZ gene in JW2500 and DS339, the zff-208::Tn10 marker gene in ME8835 was used. Because zff-208::Tn10 is linked with the rodZ gene, a P1 lysate of ME8835 was infected into JW2500 and DS339, yielding DS366 (zff-208::Tn10, rrfG) and DS368 (ispA-A164T, zff-208::Tn10, rrfG). Because yfiR is linked with the rrfG gene, ΔyfiR::kan was used as a marker gene to restore the wild-type rrfG gene in these strains. A P1 lysate of JW2584 (ΔyfiR::kan) was infected into DS366 and DS368. Elimination of the rrfG mutation from DS366 and DS368 was confirmed by DNA sequencing of the transductants, resulting in DS410 (ΔyfiR::kan, zff-208::Tn10) and DS413 (ispA-A164T, ΔyfiR::kan, zff-208::Tn10), respectively. The kan gene, which is flanked with flippase recombination targets (FRT) within yfiR of DS410 and DS413, was eliminated by a site-specific recombination system using the FRT yeast recombination targets. Plasmid pCP20 (6, 7), carrying the yeast FLP was used for the elimination. The transformed cells were incubated at 42°C to segregate pCP20, yielding DS417 (ΔyfiR, zff-208::Tn10) and DS419 (ispA-A164T, ΔyfiR, zff-208::Tn10). Finally, to reintroduce the rodZ deletion mutation, DS426 (ΔrodZ::kan ΔyfiR, zff-208::Tn10) and DS428 (ΔrodZ::kan ispA-A164T, ΔyfiR, zff-208::Tn10) were constructed by P1 transduction of JW2500 (ΔrodZ::kan), respectively.