Sinorhizobium meliloti Functionally Replaces 3-Oxoacyl-Acyl Carrier Protein Reductase (FabG) by Overexpressing NodG During Fatty Acid Synthesis

In Sinorhizobium meliloti, the nodG gene is located in the nodFEG operon of the symbiotic plasmid. Although strong sequence similarity (53% amino acid identities) between S. meliloti NodG and Escherichia coli FabG was reported in 1992, it has not been determined whether S. meliloti NodG plays a role in fatty acid synthesis. We report that expression of S. meliloti NodG restores the growth of the E. coli fabG temperature-sensitive mutant CL104 under nonpermissive conditions. Using in vitro assays, we demonstrated that NodG is able to catalyze the reduction of the 3-oxoacyl-ACP intermediates in E. coli fatty acid synthetic reaction. Moreover, although deletion of the S. meliloti nodG gene does not cause any growth defects, upon overexpression of nodG from a plasmid, the S. meliloti fabG gene encoding the canonical 3-oxoacyl-ACP reductase (OAR) can be disrupted without any effects on growth or fatty acid composition. This indicates that S. meliloti nodG encodes an OAR and can play a role in fatty acid synthesis when expressed at sufficiently high levels. Thus, a bacterium can simultaneously possess two or more OARs that can play a role in fatty acid synthesis. Our data also showed that, although SmnodG increases alfalfa nodulation efficiency, it is not essential for alfalfa nodulation.

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Isolation and Characterization of β-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Mutants of Escherichia coli and Salmonella enterica Serovar Typhimurium

Journal of Bacteriology ◽

10.1128/jb.186.6.1869-1878.2004 ◽

2004 ◽

Vol 186 (6) ◽

pp. 1869-1878 ◽

Cited By ~ 52

Author(s):

Chiou-Yan Lai ◽

John E. Cronan

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Fatty Acid Synthesis ◽

Salmonella Enterica ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Temperature Sensitive ◽

Carrier Protein ◽

Nonpermissive Temperature ◽

Serovar Typhimurium

ABSTRACT FabG, β-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The β-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.

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Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

Journal of Bacteriology ◽

10.1128/jb.188.1.287-296.2006 ◽

2006 ◽

Vol 188 (1) ◽

pp. 287-296 ◽

Cited By ~ 23

Author(s):

Nicholas R. De Lay ◽

John E. Cronan

Keyword(s):

Escherichia Coli ◽

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Lipid Synthesis ◽

Acid Synthesis ◽

Temperature Sensitive ◽

Carrier Protein ◽

Nonpermissive Temperature ◽

Overlap Extension

ABSTRACT Acyl carrier proteins (ACPs) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. Moreover, recent data indicate that the acyl carrier protein of Escherichia coli has a large protein interaction network that extends beyond lipid synthesis. Despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (acpP) that encodes ACP have been isolated. We report the isolation of three such mutants by a new approach that utilizes error-prone PCR mutagenesis, overlap extension PCR, and phage λ Red-mediated homologous recombination and that should be generally applicable. These mutants plus other experiments demonstrate that ACP function is essential for the growth of E. coli. Each of the mutants was efficiently modified with the phosphopantetheinyl moiety essential for the function of ACP in lipid synthesis, and thus lack of function at the nonpermissive temperature cannot be attributed to a lack of prosthetic group attachment. All of the mutant proteins were largely stable at the nonpermissive temperature except the A68T/N73D mutant protein. Fatty acid synthesis in strains that carried the D38V or A68T/N73D mutations was inhibited upon a shift to the nonpermissive temperature and in the latter case declined to a small percentage of the rate of the wild-type strain.

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RhlA Converts β-Hydroxyacyl-Acyl Carrier Protein Intermediates in Fatty Acid Synthesis to the β-Hydroxydecanoyl-β-Hydroxydecanoate Component of Rhamnolipids in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00080-08 ◽

2008 ◽

Vol 190 (9) ◽

pp. 3147-3154 ◽

Cited By ~ 110

Author(s):

Kun Zhu ◽

Charles O. Rock

Keyword(s):

Pseudomonas Aeruginosa ◽

Fatty Acid ◽

Fatty Acid Synthesis ◽

Fatty Acid Synthase ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Carrier Protein ◽

Molecular Ruler ◽

Necessary And Sufficient

ABSTRACT Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of β-hydroxydecanoyl-β-hydroxydecanoate from two molecules of β-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily β-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for β-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.

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Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.180.17.4596-4602.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4596-4602 ◽

Cited By ~ 54

Author(s):

Satyanarayana Subrahmanyam ◽

John E. Cronan

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

State Level ◽

Acid Synthesis ◽

The Other ◽

Carrier Protein ◽

Biosynthetic Enzyme ◽

E Coli ◽

Malonyl Coa

ABSTRACT β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.

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Insights into Acinetobacter baumannii fatty acid synthesis 3-oxoacyl-ACP reductases

Scientific Reports ◽

10.1038/s41598-021-86400-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Emily M. Cross ◽

Felise G. Adams ◽

Jack K. Waters ◽

David Aragão ◽

Bart A. Eijkelkamp ◽

...

Keyword(s):

Molecular Weight ◽

Fatty Acid ◽

Acinetobacter Baumannii ◽

High Molecular Weight ◽

Fatty Acid Synthesis ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Type I ◽

Pellicle Formation

AbstractTreatments for ‘superbug’ infections are the focus for innovative research, as drug resistance threatens human health and medical practices globally. In particular, Acinetobacter baumannii (Ab) infections are repeatedly reported as difficult to treat due to increasing antibiotic resistance. Therefore, there is increasing need to identify novel targets in the development of different antimicrobials. Of particular interest is fatty acid synthesis, vital for the formation of phospholipids, lipopolysaccharides/lipooligosaccharides, and lipoproteins of Gram-negative envelopes. The bacterial type II fatty acid synthesis (FASII) pathway is an attractive target for the development of inhibitors and is particularly favourable due to the differences from mammalian type I fatty acid synthesis. Discrete enzymes in this pathway include two reductase enzymes: 3-oxoacyl-acyl carrier protein (ACP) reductase (FabG) and enoyl-ACP reductase (FabI). Here, we investigate annotated FabG homologs, finding a low-molecular weight 3-oxoacyl-ACP reductase, as the most likely FASII FabG candidate, and high-molecular weight 3-oxoacyl-ACP reductase (HMwFabG), showing differences in structure and coenzyme preference. To date, this is the second bacterial high-molecular weight FabG structurally characterized, following FabG4 from Mycobacterium. We show that ΔAbHMwfabG is impaired for growth in nutrient rich media and pellicle formation. We also modelled a third 3-oxoacyl-ACP reductase, which we annotated as AbSDR. Despite containing residues for catalysis and the ACP coordinating motif, biochemical analyses showed limited activity against an acetoacetyl-CoA substrate in vitro. Inhibitors designed to target FabG proteins and thus prevent fatty acid synthesis may provide a platform for use against multidrug-resistant pathogens including A. baumannii.

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The commercial antimicrobial triclosan induces high levels of antibiotic tolerance in vitro and reduces antibiotic efficacy up to 100-fold in vivo

10.1101/090829 ◽

2016 ◽

Author(s):

Corey Westfall ◽

Ana Lidia Flores-Mireles ◽

John Isaac Robinson ◽

Aaron J.L. Lynch ◽

Scott Hultgren ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Acid Synthesis ◽

Consumer Products ◽

Infection Model ◽

Antibiotic Tolerance ◽

E Coli ◽

Wide Range ◽

Prophylactic Use

AbstractThe antimicrobial triclosan is used in a wide range of consumer products ranging from toothpaste, cleansers, socks, and baby toys. A bacteriostatic inhibitor of fatty acid synthesis, triclosan is extremely stable and accumulates in the environment. Approximately 75% of adults in the US have detectable levels of the compound in their urine, with a sizeable fraction of individuals (>10%) having urine concentrations equal to or greater than the minimal inhibitory concentration for Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Previous work has identified connections between defects in fatty acid synthesis and accumulation of the alarmone guanosine tetraphosphate (ppGpp), which has been repeatedly associated with antibiotic tolerance and persistence. Based on these data, we hypothesized that triclosan exposure may inadvertently drive bacteria into a state in which they are able to tolerate normally lethal concentrations of antibiotics. Here we report that clinically relevant concentrations of triclosan increased E. coli and MRSA tolerance to bactericidal antibiotics as much as 10,000 fold in vitro and reduced antibiotic efficacy up to 100-fold in a mouse urinary tract infection model. Genetic analysis indicated that triclosan-mediated antibiotic tolerance requires ppGpp synthesis, but is independent of growth. These data highlight an unexpected and certainly unintended consequence of adding high concentrations of antimicrobials in consumer products, supporting an urgent need to reevaluate the costs and benefits of the prophylactic use of triclosan and other bacteriostatic compounds.ImportanceAdded as a prophylactic to a wide range of consumer products, the fatty acid synthesis inhibitor triclosan accumulates to high levels in humans and the environment. Based on links between defects in fatty acid synthesis and accumulation of the alarmone ppGpp, we hypothesized that triclosan would render cells tolerant to bactericidal compounds due to ppGpp-mediated inhibition of biosynthetic capacity. Our data indicate that clinically relevant concentrations of triclosan induces higher tolerance of E. coli and methicillin resistant S. aureus (MRSA) to a panel of bactericidal antibiotics up to 10,000-fold. In a urinary tract infection model, mice exposed to triclosan exhibited bacterial loads ~100-fold higher in the bladder than control animals following ciprofloxacin challenge. These findings highlight an unexpected consequence of antimicrobials in consumer products and support an urgent need to reevaluate the costs and benefits of the prophylactic use of triclosan and other bacteriostatic compounds.

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