Fig. 2
Promoter characterization for expression vectors. A Soluble and total proteins of KM-1 were analyzed using 2D (a left panel) and 1D (a middle panel) gel electrophoresis. For the 1D analysis cells were collected at 24-, 48-, and 72-h incubation in SOT medium supplemented with 10% sucrose at 33 ℃ under agitation at 200 rpm (a right panel). Cells collected at 24-h incubation were used for 2D analysis. A spot at pI 5 and 20 kDa, indicated in a solid circle, is identified as an Hcp family type VI secretion system effector (Accession No. LC677173). The other spot at pI 7 and 14 kDa in a dotted circle is identified as a polyhydroxyalkanoate-associated protein (Phasin, Accession No. LC677174). B pUCpHAw_EGFP vector was constructed (Additional file 1: Fig. S3) to evaluate promoters of hcp and phasin genes and trc promoter. The three promoters, Phcp, Pphasin, and Ptrc were cloned upstream of the EGFP gene. Ptrc was cloned together with an E. coli lacIq gene. C Using EGFP as a reporter, expression levels of hcp and phasin promoters were investigated in KM-1 as well as E. coli trc promoter regulated by lacIq. Cells were cultured in SOT medium supplemented with 10% sucrose and 5 µg/mL chloramphenicol at 33 ℃ under agitation at 200 rpm. Cultivation times (in hours) are shown on the left. IPTG-induction was started at 10 h incubation with a final concentration of 1 mM, and cells were observed at 14-, 38-, and 62-h after IPTG-induction. Left and right photos in each panel are visual and fluorescent observations in the same fields. Scale bars show 1 µm. D Seven genes including zwf, phaA, and tesB were exchanged with EGFP in the pCmHAw_Phcp_EGFP vector. KM-1 harboring the vectors were cultured in SOT medium supplemented with 10% sucrose and 2.5 µg/mL chloramphenicol at 33 ℃ under agitation at 200 rpm and collected at 48-h cultivation. Expected positions of gene products, Zwf (57 kDa, sequence ID WP_010627120.1), PhaA (41 kDa, WP_010626348.1), and TesB (30 kDa, WP_010629752.1), are indicated with black arrows