To investigate this possibility, we first examined the sensitivity of the mutant cells to hyper- and hypo-osmotic conditions. As shown in Fig. 2a, the growth rate of mutants was about twofold reduced in hypoosmotic medium (LB without NaCl), whereas the effect of hyperosmotic medium (LB with 0.4 M NaCl) on mutant cells was smaller. In contrast, the

growth rate of the deletion mutants of surA that encodes a periplasmic chaperon was drastically reduced in hyperosmotic medium, but only mildly under hypo-osmotic pressure. The surA gene product is important for the synthesis of OMPs (Lazar & Kolter, 1996; Rouvière & Gross, 1996) and its mutant showed a synthetic lethal phenotype with ΔrodZ (Niba et al., 2007). Furthermore, ERK inhibitor research buy the culture of ΔrodZ mutant cells showed a sharp decline in OD600 nm when diluted with water instead of LB medium (Fig. 2b), whereas this was not observed with ΔsurA and wild-type cells. This strongly indicates that ΔrodZ cells are spheroplast like. However, this phenotype was less evident in stationary-phase cultures, which may be due to the physiological change of mureins associated with the growth stage or nutritional starvation (Goodell & Tomasz, 1980; Glauner et al., 1988). To further clarify whether peptidoglycan of the mutant cells was defective, we quantified peptidoglycan of the mutant

and wild-type cells using SLP reagent. The amount of peptidoglycan in the ΔrodZ mutant was calculated to be about 20% of the wild type (Table 2), a value well below 50% at which no detectable morphological

find more Nintedanib (BIBF 1120) change or slow growth was observed (Prats & de Pedro, 1989). This strongly indicates that the defective synthesis of peptidoglycan was the reason why the ΔrodZ mutant was very sensitive to hypo-osmotic pressure and exhibited significant cell lysis in liquid culture. The severe reduction of peptidoglycan observed with the ΔrodZ mutant was, however, less apparent in a later growth stage as in the case of the spheroplast-like phenotype described above, which seems to suggest that the ΔrodZ mutant is basically able to synthesize peptidoglycan, but is unable to coordinate it with cell growth. On the chromosome of E. coli and most of proteobacteria, rodZ is followed by ispG, an essential gene for isoprene synthesis. Because isoprene is required for the biosynthesis of peptidoglycan (Bouhss et al., 2008), the above results might support an idea that rodZ is functionally related to ispG. Therefore, we first investigated whether rodZ and ispG are transcribed together or not using lacZ fusion constructs, prodZ-1 and prodZ-2 (Fig. 3). The results showed that this was indeed the case and ispG is mostly expressed from the promoter located upstream of rodZ, although a minor transcription activity was still observed when this promoter was eliminated in prodZ-2 (Table 3).