, 1992). A feedback regulatory loop exists among AbrB, SigH, and Spo0A. During the early and mid-exponential phase of growth, the transition state regulator AbrB directly represses the synthesis of the sigma factor SigH. When activated by phosphorylation, Spo0A directly represses abrB transcription, thus relieving AbrB-mediated repression of spo0H and leading to SigH-dependent
transcription of spo0A (Strauch et al., 1990). The level and activity of Spo0A are progressively increased with time. It is generally believed that genes that play auxiliary roles in development, such as cannibalism and biofilm formation, are turned on by lower levels of activated Spo0A at an earlier stage, whereas genes that play a direct role in sporulation are turned on by higher levels of activated Spo0A at a later stage (Fujita & Losick, 2005; Fujita et al., 2005). In this report, Palbociclib we present the first genetic evidence that Spo0A is involved in controlling PHB accumulation and expression of genes for PHB biosynthesis in B. thuringiensis. Our findings have uncovered a new role for Spo0A in the regulation of stationary-phase-associated processes. The bacterial strains and plasmids used in this study are listed in Table 1. The oligonucleotides are listed in Supporting Information, Table S1. Escherichia coli and B. thuringiensis cells were grown in Luria–Bertani (LB) medium
(Sambrook & Russell, 2001) at 37 °C. Antibiotics were used at the following concentrations (μg mL−1): ampicillin, Romidepsin solubility dmso 100 (for E. coli); chloramphenicol, 8; erythromycin, 2; kanamycin, 50; and tetracycline, 25 (for B. thuringiensis). To construct plasmids pENA1, pENA2, pENA3, pENA4, pENA5, and pENA6 for gene disruption, DNA fragments carrying an internal region close to the N-terminus
of the phaC, sigB, sigH, spo0A, spo0F, or sigF genes were amplified by PCR using the primer pairs described in Table S1. After digestion with HindIII and BamHI, these DNA fragments were individually ligated into the thermosensitive plasmid pRN5101 (Fedhila et al., 2002). To construct plasmid pENA7 for deletion of the chromosomal abrB gene and replacement Methisazone with the kanamycin resistance gene (kan), a 0.33-kb DNA fragment containing a region located upstream of the abrB gene was amplified by PCR and digested with BamHI and EcoRI. After cloning of this DNA fragment into plasmid pDG780 (Guerout-Fleury et al., 1995), the resulting plasmid was restricted with BamHI and SalI to obtain a 1.8-kb DNA fragment carrying the kan gene. A 0.34-kb DNA fragment containing a region located downstream of the abrB gene was also amplified by PCR and digested with SalI and EcoRI. These two DNA fragments were then ligated together into BamHI- and EcoRI-digested plasmid pMAD (Arnaud et al., 2004). To construct plasmid pENA8 for overproduction of Spo0A in B.