To construct plasmid pENA9, a 0.25-kb DNA fragment containing the phaR promoter region was amplified by PCR. Cyclopamine After digestion with EcoRI and HindIII, the DNA fragment was inserted into pLC4 (Ray et al., 1985), which carries the xylE reporter gene. To construct plasmid pENA10, a 2-bp mutation was introduced into the −35 region of the putative σA-like promoter of phaR using a two-step PCR method (Higuchi et al., 1988). The resulting DNA fragment was digested with EcoRI plus HindIII and cloned into pLC4. Disruptions of the chromosomal phaC, sigB, sigH, spo0A, spo0F, or sigF gene of B. thuringiensis by integrations of plasmid pRN5101-derived pENA1, pENA2, pENA3, pENA4, pENA5, or pENA6, respectively,
through a Campbell-like single-crossover recombination were performed as described previously (Fedhila et al., 2002). The correct integrations were verified by PCR. Construction of the abrB deletion mutant BNA7, in which the abrB gene was replaced with the kanamycin resistance gene kan via a two-step gene replacement event, was performed according to the method described previously (Arnaud et al., 2004). The correct replacement of the chromosomal abrB sequence was verified by PCR. To construct the abrB spo0A double mutant BNA8, the pRN5101-derived plasmid pENA4 was introduced into
the abrB deletion mutant BNA7. Chromosomal integration of pENA4 through a single-crossover recombination was carried out as described above. The correct integration was verified click here by PCR. The PHB contents of B. thuringiensis cells were determined by GC as described Celastrol previously (Tseng et al., 2006) as well as using the method involving spectrophotometric determination of crotonic acid generated from digestion of PHB with concentrated sulfuric acid (Law & Slepecky, 1961). The sample preparation was carried out according to the method described in the literature (Tian et al., 2005), but with a slight modification as described previously (Chen et
al., 2009). Thin sections were examined on a JEM 2000EXII TEM (JEDL Inc.). Transformation of B. thuringiensis cells by electroporation was carried out as described previously(Bone & Ellar, 1989). Total RNA was isolated from B. thuringiensis according to the previously described method (Zuber & Losick, 1983). Northern blotting and primer extension analysis were performed using standard methods (Sambrook & Russell, 2001). An established method was used for spectrophotometric measurement of XylE (catechol 2,3-dioxygenase) activity (Ray et al., 1985). Protein concentrations were determined using the BCA protein assay method according to the instructions of the manufacturer (Pierce Biotechnology Inc.). Measurement of the PHB contents of B. thuringiensis revealed that this bacterium gradually accumulated PHB in the stationary phase after growth in LB medium (Fig. 1a). To demonstrate that the B. thuringiensis homolog of the B.