Strains and media
The strains used in this study, E. coli DH5α and BL21 (DE3) were stored in Key Laboratory of Molecular and Gene Engineering in Nanchang City. The strain was routinely grown in LB medium (5 g yeast extracts/l, 10 g peptone/l, and 10 g NaCl/l) at 37 °C with oxygen incubation. For anaerobic incubation, LB medium was prepared per 600-ml anaerobic bottle and sterilized under a strictly anaerobic H2 and CO2 atmosphere (80:20).
The DNA fragment encoding orfH79 was amplified from the CMS-HL rice by PCR using a primer set (forward, 5′ GCCGGATCCATGACAAATCTG CTCCGATGGCTC-3′; reverse, 5′ GCCCTCGAGTTACTTAGGAAAGACTACA CG-3′). The orfH79 gene was ligated to the pET-28a vector (Invitrogen) digested with NcoI and XholI to construct the plasmid pET-28a-orfH79. The BL21 (DE3) strains were transformed with plasmids pET-28a-orfH79 and pET-28a empty vector. As a control in some experiments, we also used pET-28a vector expressed a disulfide isomerase-like protein (PDI) gene, MTH1745, which contained a hygrophobic transmembrane structure from 10 to 30 amino acids in this protein (Ding et al. 2008).
Preparation of cell fraction
Crude membrane versus cytoplasmic cell fraction were prepared described as Arockiasamy and Krishnaswamy (2000) with some modifications. 100 ml of bacterial cell culture were harvested and centrifuged at 10,000×g for 5 min at 4 °C. The precipitation was washed twice with phosphate buffered saline (PBS), and resuspended in the same buffer containing 0.1 M PMSF and 0.1 M PI (both Roche Diagnostics). The suspension was ultrasonicated on ice for 15 min (5 s on with 5 s intervals). Cell debris and insoluble proteins were recovered by centrifugation for 1 h at 10,000×g, and the supernatant was centrifuged at 30,000×g for 2 h at 4 °C. The supernatant was retained for further analysis and the pellet containing the crude membrane was resuspended in 200 µl dilution buffer (66 mM Tris–Cl, pH 6.8, 2 % v/v 2-mercaptoethanol, 2 % SDS).
Antibodies and western blot analysis
Equal amounts of protein from the membrane and cytoplasm were separated by 18 % SDS-PAGE gel at 4 °C, and then transferred onto an Immobilon-PSQ transfer membrane (PVDF type; Millipore) at 80 V for 40 min. The membrane was incubated in 5 % w/v non-fat milk, 0.05 % v/v Tween-20, in PBS for 1 h, washed for 10 min three times in PBST (PBS, 0.05 %v/v Tween-20), and incubated in a 1:5000 dilution of mouse antiserum anti-ORFH79 overnight at 4 °C. After four washes with PBST, the membrane was incubated with rabbit anti-mouse IgG conjugated with alkaline phosphatase (AP) in PBST solution for 2 h. After four 10-min washes in PBST, the signal was visualized by chemiluminescent detection (Pierce, Rockford Rockford, IL) according to the manufacturer’s protocol. Antibody against ORFH79 was a gift from professor Shaoqing Li, College of Life Science, Wuhan University.
Escherichia coli growth curve assay
The growth curve assay was performed to ascertain the effect of expressing OrfH79 in E. coli cells were transformed with either pET-28a-orfH79 or pET-28a. The transformants were incubated in LB medium at 37 °C overnight. For oxygen incubation, 30 ml LB medium supplemented with 30 µl preculture was incubated at 37 °C, with shaking. At an OD600 of 0.6, the culture was separated into two equal subcultures. IPTG was added to one of the cultures, at a final concentration of 1 mM. The cultures were grown at 37 °C with shaking (250 rpm), and the cell density (OD600) of these culture were monitored by withdrawing aliquots at various times. For anaerobic incubation, 600 µl overnight preculture was added to LB medium prepared per 600-ml anaerobic bottle and autoclave under a strictly anaerobic H2 and CO2 atmosphere (80:20), and incubated at 37 °C, with shaking. At an OD600 of 0.3, the culture was separated into two equal subcultures. IPTG was added to one culture, at a final concentration of 1 mM. The cultures were grown at 37 °C with shaking (250 rpm), and the cell density (OD600) of these culture were monitored by withdrawing aliquots at various times.
BL21(DE3) pLYsS cells containing pET-28a-orfH79 and pET-28a were cultured in 5 ml LB medium supplemented with antibiotics at 37 °C. Each preculture was diluted 1:1000 in fresh LB medium containing antibiotics. The resulting culture was incubated at 37 °C until the cells reached the early exponential growth phase. Each culture was then divided into two subcultures. One subculture was induced with 1 mM IPTG whereas the other was used as a control. Both subcultures were incubated at 37 °C, with shaking, for an additional 2 h. Cellular respiration was measured at HPES-KPR buffer (50 mM HPES, pH7.4, 100 mM NaCL, 5 mM KCL, 1 mM MgCL2, 1 mM NaH2PO4, 1 mM d-glucose and 1 mM CaCl2). Respiration was measured using the temperature-controlled chamber of a chlorolab 2 electrode (Hansatech, UK) containing 2 ml of buffer. The respiration rate was measured by the detection of oxygen consumption at 37 °C.
Membrane observation of E. coli by scanning electron microscopeye
The bacterial cells were fixed with 2.5 % glutaradehyde overnight at 4 °C. The fixed cells were washed three times with phosphate buffer (0.1 M, pH 7.0), 15 min each time. The bacterial cells were dehydrated in ascending concentration of ethanol (10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 100 and 100 %) at 15 min exposure for each concentration. The bacterial cells were further dehydrated in different ratios of ethanol: acetone (3:1, 1;1 and 1:3) for 20 min for each mixture and then washed with pure acetone four times each for 20 min. These bacterial cells were subjected to critical point drying using liquid CO2 and the cells mounted on a stub. These cells were coated with gold and examined by using scanning electron microscope.
Detection of pyruvate kinase and α-ketoglutarate dehydrogenase activity
BL21(DE3) pLYsS cells, harboring the expression vectors pET-28a-orfH79 and pET-28a, were cultured in LB medium until the optical density reached 0.6–0.8. Each culture was then divided into two subcultures. One subculture was induced with 1 mM IPTG whereas the other was used as a control. Cells were harvested at 6 h and 12 h, after induction with IPTG. Cells were disrupted by sonication and centrifuged to remove cell debris. The supernatants were used to determine the protein quantity and for use in enzymatic assays. Pyruvate kinase activity was measured using the ultraviolet chromatometry method following the protocol of the pyruvate kinase activity assay kit (Nanjing Jiancheng, China). The α-ketoglutarate dehydrogenase activity determination was performed described by Flora (Pettit et al. 1973).
All experimental data are the mean of at least three independent replicates, and comparisions between transformants were performed using one way ANOVA with Duncan’s multiple range test. All the statistical analyses were performed using SPSS software.