, 2009; Bendezúet al., 2009). The rodZ gene codes for an inner membrane protein that possesses a putative DNA-binding domain of a helix-turn-helix (HTH) motif. Furthermore, RodZ was shown to interact with bacterial actin MreB (Bendezúet al., 2009; van den Ent
Protein Tyrosine Kinase inhibitor et al., 2010), which may determine the place of cell wall synthesis (Alyahya et al., 2009; White et al., 2010). However, the exact role of RodZ in cell-shape determination remains to be elucidated. Mutants of rodZ were found to be growth deficient and exhibited a spherical form instead of the normal rod shape. In this work, we have further characterized the ΔrodZ mutant as well as its pseudorevertant and present evidence that strongly indicates the involvement of rodZ in the biosynthesis of peptidoglycans. All E. coli strains used were derivatives of KR0401 (Niba et al., 2007). Deletion mutations ΔrodZ∷kan and ΔsurA∷kan were introduced from mutants of each gene in the Keio collection (Baba et al., 2006) by P1kc-mediated transduction. lacZ fusions for promoter
analysis were constructed as follows: from approximately 500 bp upstream of the first gene of each operon together with approximately 12 bp from the translational start site inside the ORF was PCR-amplified and cloned into pJL28 or pJL30 protein-fusion vectors (Lucht et al., 1994). check details In the case of the flhB gene, the region from nucleotide −7 to +5 was replaced with that of fliE, because, for unknown reasons, no β-galactosidase activity was obtained with its own initiator codon. pJL28-fliA (pMW198) and pJL29-flgB (pMW211) click here have been described (Lehnen et al.,
2002). Single-copy (chromosomal) lacZ fusion strains were subsequently obtained from plasmid-bearing strains via phage λInCh as described (Boyd et al., 2000). β-Galactosidase activity was measured according to Miller (1972). The plasmid vector for the expression of the cloned gene with the C-terminal S-tag, pBADs, was constructed based on pBAD322 (Cronan, 2006) by replacing the SphI–HindIII region with a 520-bp fragment amplified from pSHLeu (Gan et al., 2002). ΔHTH and Δ(30-133) deletions were introduced using a mutagenesis method of overlap extension reported by Warrens et al. (1997). Cells were grown in Luria–Bertani (LB) medium at 37 °C to the exponential growth phase. Formvar-carbon-coated copper grids were floated for 3 min onto a drop of cell culture, washed on drops of 0.9% NaCl and distilled water and then stained with 1% uranyl acetate. Images were obtained by a transmission electron microscope (H-7100, Hitachi, Tokyo, Japan) at an acceleration voltage of 75 kV. Peptidoglycan was isolated essentially as described (Hervéet al., 2007). Cells from 200-mL LB cultures grown at 37 °C to an OD600 nm of ca. 0.75 were used. After lysis of cells by the addition of hot 4% sodium dodecyl sulfate solution, followed by overnight incubation at room temperature, a cell wall fraction was obtained by centrifugation at 100 000 g for 1 h, washed with water and suspended in 0.