Response of Bacillus subtilis to Cerulenin and Acquisition of Resistance

ABSTRACT Cerulenin is a fungal mycotoxin that potently inhibits fatty acid synthesis by covalent modification of the active site thiol of the chain-elongation subtypes of β-ketoacyl-acyl carrier protein (ACP) synthases. The Bacillus subtilis fabF (yjaY) gene (fabFb ) encodes an enzyme that catalyzes the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. There were two mechanisms by which B. subtilis adapted to exposure to this antibiotic. First, reporter gene analysis demonstrated that transcription of the operon containing the fabF gene increased eightfold in response to a cerulenin challenge. This response was selective for the inhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-ACP reductase, triggered an increase in fabF reporter gene expression while nalidixic acid did not. Second, spontaneous mutants arose that exhibited a 10-fold increase in the MIC of cerulenin. The mutation mapped at the B. subtilis fabF locus, and sequence analysis of the mutant fabF allele showed that a single base change resulted in the synthesis of FabFb[I108F]. The purified FabFb and FabFb[I108F] proteins had similar specific activities with myristoyl-ACP as the substrate. FabFb exhibited a 50% inhibitory concentration (IC50) of cerulenin of 0.1 μM, whereas the IC50 for FabFb[I108] was 50-fold higher (5 μM). These biochemical data explain the absence of an overt growth defect coupled with the cerulenin resistance phenotype of the mutant strain.

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Subinhibitory cerulenin inhibits staphylococcal exoprotein production by blocking transcription rather than by blocking secretion

Microbiology ◽

10.1099/mic.0.28102-0 ◽

2005 ◽

Vol 151 (9) ◽

pp. 3059-3069 ◽

Cited By ~ 14

Author(s):

Rajan P. Adhikari ◽

Richard P. Novick

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Covalent Modification ◽

Extracellular Proteins ◽

Chain Elongation ◽

Northern Blot Hybridization ◽

Normal Fatty Acid ◽

Membrane Function

Cerulenin is an antibiotic that inhibits fatty acid synthesis by covalent modification of the active thiol of the chain-elongation subtypes of β-ketoacyl-acyl carrier protein synthase. It also inhibits other processes that utilize essential thiols. Cerulenin has been widely reported to block protein secretion at sub-MIC levels, an effect that has been postulated to represent interference with membrane function through interference with normal fatty acid synthesis. This study confirms the profound reduction in extracellular proteins caused by low concentrations of the antibiotic, and shows by Northern blot hybridization that this reduction is due to interference with transcription. By exchanging promoters between entB, a gene that is inhibited by cerulenin, and entA, a gene that is not, it was also shown that the antibiotic does not block secretion. Subinhibitory concentrations of cerulenin were also found to block transcriptional activation of at least two regulatory determinants, agr and sae, that function by signal transduction. Interference with the activation of these and other regulatory determinants probably accounts for much of the inhibitory effect on exoprotein production of sub-MIC concentrations of cerulenin.

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Divergent acyl carrier protein decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis in malaria parasites

eLife ◽

10.7554/elife.71636 ◽

2021 ◽

Vol 10 ◽

Author(s):

Seyi Falekun ◽

Jaime Sepulveda ◽

Yasaman Jami-Alahmadi ◽

Hahnbeom Park ◽

James A Wohlschlegel ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Acyl Chain ◽

Acid Synthesis ◽

Complex Iii ◽

Carrier Protein ◽

Prosthetic Group ◽

Malaria Parasites ◽

Mitochondrial Fatty Acid

Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis and biochemically couple FASII activity to mitochondrial electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast, the mitochondrion of Plasmodium falciparum malaria parasites lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other apicomplexan pathogens, including Toxoplasma and Babesia. This discovery unveils an evolutionary driving force to retain interaction of mitochondrial Fe-S cluster biogenesis with ACP independent of its eponymous function in FASII.

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The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

eLife ◽

10.7554/elife.17828 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 76

Author(s):

Jonathan G Van Vranken ◽

Mi-Young Jeong ◽

Peng Wei ◽

Yu-Chan Chen ◽

Steven P Gygi ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Acyl Chain ◽

Acid Synthesis ◽

Carrier Protein ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Mitochondrial Fatty Acid

Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis.

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Acquisition of exogenous fatty acids renders apicoplast-based biosynthesis dispensable in tachyzoites of Toxoplasma

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013004 ◽

2020 ◽

Vol 295 (22) ◽

pp. 7743-7752 ◽

Cited By ~ 3

Author(s):

Xiaohan Liang ◽

Jianmin Cui ◽

Xuke Yang ◽

Ningbo Xia ◽

Yaqiong Li ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Fatty Acid Synthesis ◽

Pyruvate Dehydrogenase ◽

Acyl Carrier Protein ◽

Pyruvate Dehydrogenase Complex ◽

Acid Synthesis ◽

Lytic Cycle ◽

Growth Defect ◽

Wide Range

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9–assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo. Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

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In Vivo Evidence that S-Adenosylmethionine and Fatty Acid Synthesis Intermediates Are the Substrates for the LuxI Family of Autoinducer Synthases

Journal of Bacteriology ◽

10.1128/jb.180.10.2644-2651.1998 ◽

1998 ◽

Vol 180 (10) ◽

pp. 2644-2651 ◽

Cited By ~ 86

Author(s):

Dale L. Val ◽

John E. Cronan

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Acyl Chain ◽

Acid Synthesis ◽

Homoserine Lactone ◽

Fatty Acid Degradation ◽

Acid Degradation ◽

Acyl Coenzyme A

ABSTRACT Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis ofN-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in β-oxidative fatty acid degradation. Also, conditions known to induce the fadregulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on β-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellularS-adenosylmethionine caused by expression of bacteriophage T3 S-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from S-adenosylmethionine.

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