3) and introducing them into the ΔrodZ mutant and wild type. The β-galactosidase activity of prodZ-3, prodZ-1-ΔHTH and prodZ-1-Δ(30-133) was 1.6, 1.5 and 3.4-fold higher, respectively, in the ΔrodZ mutant. In wild-type cells, however, the expression of ispG was decreased about 50% when rodZ on the plasmid was partially deleted, which might indicate that the RodZ protein is required for the coordinated synthesis of rodZ and ispG. Interestingly,

the expression of ispG from prodZ-1-ΔHTH was reduced in the rodZ mutant, although to a lesser extent compared with the wild type, indicating that the RodZ lacking the HTH domain might partially retain its function. Also, the ΔrodZ mutant carrying this plasmid grew slightly faster. Finally, in order to locate the minor promoter(s) Idasanutlin purchase observed with prodZ-2, we constructed additional lacZ fusions (Fig. 3) and examined their β-galactosidase activity (Table 3). The results showed that, indeed, a promoter(s) existed within Deforolimus price the rodZ-orf as well as in the intergenic region, both of which showed higher activity in the ΔrodZ mutant compared with the wild type, while the expression of ispE, another gene involved in isoprene synthesis and located in a different operon, was not increased, suggesting that

the effect of RodZ is specific to the expression of the rodZ-ispG operon. It seems that a balanced expression of some sorts between rodZ and ispG might be important, although we were unable to explain these results in an unequivocal manner. During the analysis described here, we often encountered inconsistent results with the ΔrodZ mutant and noticed that derivatives that were motile and grew faster emerged spontaneously within the population. By PCR analysis,

we confirmed the presence of the ΔrodZ (rodZ∷kan) mutation in those faster-growing derivatives (data not shown). Subsequently, we isolated one such pseudorevertant, termed KR0401ΔrodZ-mot+, and characterized the phenotype. The cells grew and expressed fliA and fliC at a level similar to that of Cytidine deaminase the wild type (Table 1). The cell shape was almost rod type, although more irregular and asymmetrical compared with the wild type (Fig. 1i). The cells tended to be more elongated than the wild type in contrast to the original ΔrodZ mutant. When extra copies of rodZ were introduced, some cells showed a filamentous morphology (Fig. 1j). The amount of peptidoglycan was also significantly higher than the original ΔrodZ mutant (Table 2). Furthermore, the expression of the plasmid-borne ispG measured by the fused lacZ activity was decreased as in the wild type when either the ΔHTH or the Δ(6-30-133) deletion was introduced (Fig. 3, Table 3).