BackgroundFatty acids synthesized in chloroplast are transported to endoplasmic reticulum (ER) for triacylglycerols (TAGs) resembling. The development of chloroplast also requires lipids trafficking from ER to chloroplast. The membrane contact sites (MCSs) between ER and chloroplast has been demonstrated to be involved for the trafficking of lipids and proteins. Lipids trafficking between ER and chloroplast is often accompanied by lipids interconversion. However, it is rarely known how lipids interconversion happens during their trafficking.Methodology/Principal FindingsWe cloned a lipase gene from Brassica napus L., designated as BnCLIP1. Green fluorescence protein (GFP)-tagged BnCLIP1 was shown to locate at the MCSs between ER and chloroplasts in tobacco leaves. Heterogeneous expression of BnCLIP1 in Saccharomyces cerevisiae (pep4) reduced the total amount of fatty acid. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the truncated BnCLIP1 had a substrate preference for C16:0 lipids in Saccharomyces cerevisiae (pep4). To probe the physiological function of BnCLIP1, two Brassica napus lines with different oil-content were introduced to investigate the transcript patterns of BnCLIP1 during seed development. Intriguingly, the transcript level of BnCLIP1 was found to be immediately up-regulated during the natural seed senescence of both lines; the transcription response of BnCLIP1 in the high oil-content seeds was faster than the lower ones, suggesting a potential role of BnCLIP1 in affecting seed oil synthesis via regulating chloroplast integrity. Further researches showed that chemical disruption of leaf chloroplast also activated the transcription of BnCLIP1.Conclusions/SignificanceThe findings of this study show that BnCLIP1 encodes a lipase, localizes at the MCSs and involves in chloroplast development.

Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and polyhydroxyalkanoic acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty acids of 2-bromohexanoic acid (2-BrHA), 2-bromooctanoic acid (2-BrOA) and 2-bromodecanoic acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa ΔphaG and ΔrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA (≥90% at 2 mM) > 2-BrOA > 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets.

Several factors including the culture temperature, bivalent ion, and osmotic stress were gradually optimized for preparing efficient competent cells. The effect of culture temperature on the transformation efficiency (TrE) of DH5α was tested with 100 mM CaCl. The lower culture temperature at 18 °C resulted in higher TrE of 2.5 × 10 cfu/μg, which was about 3.5 times of that obtained at 37 °C. Bivalent ions including Ca, Mn, Mg, and Ni were tested independently or combinatorially at a total concentration of 100 mM. Ni showed a significantly inhibition on the TrE, and various concentration combinations of Ca, Mg, and Mn were tested. The TrE was improved up to 1.8 ± 0.4 × 10 cfu/μg, when a combination of 25 mM Ca, 50 mM Mg, and 25 mM Mn was applied. Further supplement of 0.8% (w/v) PEG lead to a slight decrease in the TrE, whereas supplement of 25 mM sucrose contributed to another increase in the TrE by 17% up to 2.1 ± 0.3 × 10 cfu/μg. These results indicated that the culture temperature and bivalent ion were important factors affecting the TrE of A chemical method for preparing efficient competent cells of was provided.

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