A total synthesis of the cyclic lipodepsipeptide natural product orfamide A was achieved. By developing a synthesis format using an aminoacid ester building block and SPPS protocol adaptation, a focused library of target compounds was obtained, in high yield and purity. Spectral and LC-HRMS data of all library members with the isolated natural product identified the 5 Leu residue to be d-and the 3'-OH group to be R-configured. The structural correction of orfamide A by chemical synthesis and analysis was confirmed by biological activity comparison in Chlamydomonas reinhardtii, which indicated compound configuration to be important for bioactivity. Acute toxicity was also found against Trypanosoma brucei, the parasite causing African sleeping sickness.
The unicellular alga Chlamydomonas reinhardtii and the bacterium Pseudomonas protegens serve as a model to study the interactions between photosynthetic and heterotrophic microorganisms. P. protegens secretes the cyclic lipopeptide orfamide A that interferes with cytosolic Ca 2+ homeostasis in C. reinhardtii resulting in deflagellation of the algal cells. Here, we studied the roles of additional secondary metabolites secreted by P. protegens using individual compounds and co-cultivation of algae with bacterial mutants. Rhizoxin S2, pyrrolnitrin, pyoluteorin, 2,4-diacetylphloroglucinol (DAPG) and orfamide A all induce changes in cell morphology and inhibit the growth of C. reinhardtii. Rhizoxin S2 exerts the strongest growth inhibition, and its action depends on the spatial structure of the environment (agar versus liquid culture). Algal motility is unaffected by rhizoxin S2 and is most potently inhibited by orfamide A (IC 50 = 4.1 μM). Pyrrolnitrin and pyoluteorin both interfere with algal cytosolic Ca 2+ homeostasis and motility whereas high concentrations of DAPG immobilize C. reinhardtii without deflagellation or disturbance of Ca 2+ homeostasis. Co-cultivation with a regulatory mutant of bacterial secondary metabolism (ΔgacA) promotes algal growth under spatially structured conditions. Our results reveal how a single soil bacterium uses an arsenal of secreted antialgal compounds with complementary and partially overlapping activities.
The antagonistic bacterium Pseudomonas protegens secretes the cyclic lipopeptide orfamide A, which triggers a Ca2+ signal, causing the deflagellation of the green microalga Chlamydomonas reinhardtii. By investigating targeted synthetic orfamide A variants and inhibitors, we found that at least two Ca2+-signalling pathways and TRP channels are involved in this response.
The effect of the addition of high-octane oxygenated fuel including methyl tertiary butyl ether (MTBE), ethanol, and methanol on combustion phasing and combustion rate of homogeneous charge compression ignition (HCCI) combustion fueled with n-heptane as base fuel was comparatively investigated. In this paper, n-heptane was blended with MTBE, ethanol, and methanol from 10 vol % to 60 vol % at 10 vol % increments, respectively. Three kinds of blended fuels were injected into intake air, and their premixed HCCI combustions were studied comparatively. The results show that the HCCI combustion fueled with high-octane oxygenated fuel/n-heptane blended fuels is delayed in the combustion phasing and suppressed in combustion rate in contrast to that with pure n-heptane. Given the higher fuel/air equivalence ratio, the combustion phasing of blended fuels is progressively delayed with the increasing proportion of MTBE, ethanol, or methanol, so that the high-temperature heat release (HTHR) can occur near top-dead center (TDC) or later; at the same time, the HCCI combustion can be suppressed to partial burn or even misfire when the proportion of MTBE, ethanol, and methanol is increased to some extent, respectively. On the other hand, MTBE, ethanol, and methanol have some differences in how they affect the n-heptane-fueled HCCI combustion. In view of the capability of delaying combustion phasing, methanol is exhibited as the most powerful of the three fuels and ethanol is stronger than MTBE. In view of suppressing the combustion rate of HCCI combustion, methanol is the most effective and MTBE is the least powerful among the three fuels. When total fueling rate is kept at 18.7 mg/cycle, the misfire of methanol/n-heptane blended fuels occurs near methanol 40 vol % (M40), and that of ethanol/n-heptane blended fuels occurs near ethanol 50 vol % (E50); however, MTBE/n-heptane blended fuels do not misfire till MTBE 60 vol % (MTBE60). The differences among the three fuels fundamentally result from their performance of difficult autoignition. At the same time, the oxygen content in the high-octane oxygenated fuels may improve the HTHR of HCCI combustion that is still suppressed by the addition of these fuels. On the basis of the difference of three high-octane oxygenated fuels in the control of combustion phasing and combustion rate, it is noted that MTBE has more advantages over methanol and ethanol in the potential of extending the operating range of n-heptane-fueled HCCI combustion by adjusting the addition of MTBE, ethanol, and methanol, respectively, at the same total fueling rate.
Summary The antagonistic bacterium Pseudomonas protegens secretes the cyclic lipopeptide (CLiP) orfamide A, which triggers a Ca2+ signal causing rapid deflagellation of the microalga Chlamydomonas reinhardtii. We performed chemical synthesis of orfamide A derivatives and used an aequorin reporter line to measure their Ca2+ responses. Immobilization of algae was studied using a modulator and mutants of transient receptor potential (TRP)‐type channels. By investigating targeted synthetic orfamide A derivatives, we found that N‐terminal amino acids of the linear part and the terminal fatty acid region are important for the specificity of the Ca2+‐signal causing deflagellation. Molecular editing indicates that at least two distinct Ca2+‐signaling pathways are triggered. One is involved in deflagellation (Thr3 change, fatty acid tail shortened by 4C), whereas the other still causes an increase in cytosolic Ca2+ in the algal cells, but does not cause substantial deflagellation (Leu1 change, fatty acid hydroxylation, fatty acid changes by 2C). Using mutants, we define four TRP‐type channels that are involved in orfamide A signaling; only one (ADF1) responds additionally to low pH. These results suggest that the linear part of the CLiP plays one major role in Ca2+ signaling, and that orfamide A uses a network of algal TRP‐type channels for deflagellation.
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