A two-helix motif positions the lysophosphatidic acid acyltransferase active site for catalysis within the membrane bilayer

Robertson, R.M.; Yao, Jiangwei; Gajewski, Stefan; Kumar, Gyanendra; Martin, Erik W.; Rock, Charles O.; White, Stephen W.

doi:10.1038/nsmb.3436

Cited by 56 publications

(83 citation statements)

References 56 publications

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“…The pPJ131 vector contains an amino‐terminal His 6 ‐tag that was cloned in frame with each gene using the NdeI restriction site. The p Ec PlsC vector (Robertson et al, ), p Ec FadD vector (Yao et al, ), and p Ng Aas vector (Yao et al, ) were described previously. Af Aas1 and Af Aas2 were cloned into the pET28b vector (Novagen) using the 5′‐NdeI and 3′‐EcoRI restriction sites to introduce an amino‐terminal His 6 ‐tag.…”

Section: Methodsmentioning

confidence: 99%

“…Overnight cultures of strain SM2‐1 ( plsC (Ts)) harboring the PlsC expression plasmids were grown at 30°C, subcultured into Luria broth with and without exogenous 18:1(Δ9), and grown at 42°C to A 600 = 0.8. Lipids were extracted via the Bligh and Dyer method (Bligh & Dyer, ) from each experiment, and PE molecular species composition was determined (Robertson et al, ).…”

Section: Methodsmentioning

confidence: 99%

“…The distribution of fatty acids between the 1‐ and 2‐positions of A. finegoldii PE was determined by radiolabeling cultures with [ 14 C]acetate or [ 14 C]oleate, and purifying labeled PE from the lipid extracts by thin‐layer chromatography on Silica Gel H plates. Silica‐bound [ 14 C]PE was digested with Naja mossambica mossambica snake venom phospholipase A 2 to deacylate the 2‐position fatty acid as described (Robertson et al, ). After 3 hr at room temperature, lipids were extracted via the Bligh and Dyer method (Bligh & Dyer, ) and separated on a Silica Gel H plate (Analtech, Inc.) developed with chloroform:methanol:ammonium hydroxide (80/25/4, v/v), and the distribution of radioactivity on the plate was determined using a Bioscan Imaging detector.…”

Section: Methodsmentioning

confidence: 99%

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Fatty acid activation and utilization by Alistipes finegoldii, a representative Bacteroidetes resident of the human gut microbiome

Radka

Frank

Rock

et al. 2020

Molecular Microbiology

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Members of the Bacteroidetes phylum, represented by Alistipes finegoldii, are prominent anerobic, Gram‐negative inhabitants of the gut microbiome. The lipid biosynthetic pathways were analyzed using bioinformatic analyses, lipidomics, metabolic labeling and biochemistry to characterize exogenous fatty acid metabolism. A. finegoldii only produced the saturated fatty acids. The most abundant lipids were phosphatidylethanolamine (PE) and sulfonolipid (SL). Neither phosphatidylglycerol nor cardiolipin are present. PE synthesis is initiated by the PlsX/PlsY/PlsC pathway, whereas the SL pathway is related to sphingolipid biosynthesis. A. finegoldii incorporated medium‐chain fatty acids (≤14 carbons) into PE and SL after their elongation, whereas long‐chain fatty acids (≥16 carbons) were not elongated. Fatty acids >16 carbons were primarily incorporated into the 2‐position of phosphatidylethanolamine at the PlsC step, the only biosynthetic enzyme that utilizes long‐chain acyl‐ACP. The ability to assimilate a broad‐spectrum of fatty acid chain lengths present in the gut environment is due to the expression of two acyl‐acyl carrier protein (ACP) synthetases. Acyl‐ACP synthetase 1 had a substrate preference for medium‐chain fatty acids and synthetase 2 had a substrate preference for long‐chain fatty acids. This unique combination of synthetases allows A. finegoldii to utilize both the medium‐ and long‐chain fatty acid nutrients available in the gut environment to assemble its membrane lipids.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Fatty acid activation and utilization by Alistipes finegoldii, a representative Bacteroidetes resident of the human gut microbiome

Radka

Frank

Rock

et al. 2020

Molecular Microbiology

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“…We did not experimentally confirm whether SM43 PlsC catalyzes acylation of both LPA and LPC; however, we hypothesize this to be the case. The only crystal structure available for PlsC is from the hyperthermophiliic bacterium Thermotoga maritima (67), with which the SM43 PlsC shares only weak (24%) amino acid sequence similarity. Biochemical studies of purified SM43 PlsC and structural modeling of this enzyme in comparison with PlsC from other bacteria will be required to understand the mechanistic basis for SM43 PlsC acylation of LPC.…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylcholine biosynthesis in Mitis group streptococci via host metabolite scavenging

Joyce¹,

Guan

Palmer³

2019

Preprint

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14The Mitis group streptococci include the major human pathogen Streptococcus 15 pneumoniae and the oral opportunistic pathogens S. mitis and S. oralis which are 16 primary colonizers in the oral cavity and agents of bacteremia and infective endocarditis 17 in immunocompromised patients. Bacterial membrane lipids play crucial roles in 18 microbe-host interactions, yet for many pathogens, the composition of the membrane is 19 poorly understood. In this study, we characterized the lipidomes of selected species of 20 Mitis group streptococci and investigated the mechanistic basis for biosynthesis of the 21 phospholipid phosphatidylcholine (PC). PC is a major lipid in eukaryotic cellular 22 membranes, but it is considered to be comparatively rare in bacterial taxa. Using liquid 23 chromatography/mass spectrometry (LC/MS) in conjunction with stable isotope tracing, 24 we determined that Mitis group streptococci synthesize PC via the rare host metabolite 25 scavenging pathway, the glycerophosphocholine (GPC) pathway, which is largely 26 uncharacterized in bacteria. Furthermore, we identify the acyltransferase, PlsC, as 27 playing a major role in the pathway by acylating lysoPC to PC. Our work demonstrates 28 that Mitis group streptococci including S. pneumoniae remodel their membrane in 29 response to the major human metabolites GPC and lysoPC. 30 31 Key words 32 33 Phosphatidylcholine, phospholipid, Streptococcus pneumoniae, Streptococcus mitis, 34 Streptococcus oralis, Mitis group streptococci, glycerophosphocholine 35 3 36 Significance 37 38We lack fundamental information about the structure of the cellular membrane even for 39 the best studied pathogens of critical significance for human health. The Mitis group 40 streptococci are closely linked to humans in health and disease. Here, we demonstrate 41 that these streptococci scavenge major human metabolites and use them to synthesize 42 the membrane phospholipid phosphatidylcholine. Our work is significant because it 43 identifies a mechanism by which the major human pathogen S. pneumoniae and the 44 primary human oral colonizers S. mitis and S. oralis remodel their membrane in 45 response to the host.46 48 The Mitis group streptococci are Gram-positive bacteria that natively inhabit the human 49 oral cavity, nasopharynx, and gastrointestinal tract (1). They include the species S. mitis 50 and S. oralis, which are among the first colonizers of the human oral cavity from birth 51 and are believed to play important roles in host-microbe-microbe interactions by 52 creating an anchor for biofilm formation with other oral microbiota (2, 3). They are also 53 opportunistic pathogens causing bacteremia and infective endocarditis (4-7). They also 54 include the major human pathogen, S. pneumoniae. S. pneumoniae has >99% 16S 55 rRNA sequence identity with S. mitis and S. oralis (8-10), and with which they 56 exchange capsule biosynthesis and antibiotic resistance genes (11, 12) and show 57 antibody cross-reactivity (13). 58 59 We, and others, recently reported that Mit...

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“…L48 is close to the HXXXXD motif, which is part the active site of the enzyme. Similarly, A61 and P110 are in very close proximity to the active center of the enzyme [68]…”

Section: A Reduction Of Phospholipid Biosynthesis Helps To Overcome Tmentioning

confidence: 99%

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

Krüger

Herzberg

Rath

et al. 2020

Preprint

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In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis . In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA ( A mino acid im porter A , formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis . Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) suggesting the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

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A two-helix motif positions the lysophosphatidic acid acyltransferase active site for catalysis within the membrane bilayer

Cited by 56 publications

References 56 publications

Fatty acid activation and utilization by Alistipes finegoldii, a representative Bacteroidetes resident of the human gut microbiome

Fatty acid activation and utilization by Alistipes finegoldii, a representative Bacteroidetes resident of the human gut microbiome

Phosphatidylcholine biosynthesis in Mitis group streptococci via host metabolite scavenging

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

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