Electrospray ionization mass spectrometric analysis of phospholipids of <i>Escherichia coli</i>

Sweetman, Gavain; Trinei, Mirella; Modha, J.; Kusel, J. R.; Freestone, Primrose; Fishov, Itzhak; Joseleau‐Petit, Danièle; Redman, C.; Farmer, Peter B.; Norris, Victor

doi:10.1111/j.1365-2958.1996.tb02504.x

Cited by 29 publications

(28 citation statements)

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“…We therefore conclude that the integrity of the protein is crucial to the stability of these structures. To confirm the presence or absence of E. coli lipid within the proteinosomes, we analyzed them by means of electrospray ionization MS. No species were detected with the molecular mass that corresponded to lipids typically associated with the E. coli cell membrane (22) (Fig. S6), suggesting that the vesicles are pure protein complexes rather than protein-lipid assemblies.…”

Section: Resultsmentioning

confidence: 99%

Self-assembled MmsF proteinosomes control magnetite nanoparticle formation in vitro

Rawlings

Bramble

Walker

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Magnetotactic bacteria synthesize highly uniform intracellular magnetite nanoparticles through the action of several key biomineralization proteins. These proteins are present in a unique lipidbound organelle (the magnetosome) that functions as a nanosized reactor in which the particle is formed. A master regulator protein of nanoparticle formation, magnetosome membrane specific F (MmsF), was recently discovered. This predicted integral membrane protein is essential for controlling the monodispersity of the nanoparticles in Magnetospirillum magneticum strain AMB-1. Two MmsF homologs sharing over 60% sequence identity, but showing no apparent impact on particle formation, were also identified in the same organism. We have cloned, expressed, and used these three purified proteins as additives in synthetic magnetite precipitation reactions. Remarkably, these predominantly α-helical membrane spanning proteins are unusually highly stable and water-soluble because they self-assemble into spherical aggregates with an average diameter of 36 nm. The MmsF assembly appears to be responsible for a profound level of control over particle size and iron oxide (magnetite) homogeneity in chemical precipitation reactions, consistent with its indicated role in vivo. The assemblies of its two homologous proteins produce imprecise various iron oxide materials, which is a striking difference for proteins that are so similar to MmsF both in sequence and hierarchical structure. These findings show MmsF is a significant, previously undiscovered, protein additive for precision magnetite nanoparticle production. Furthermore, the self-assembly of these proteins into discrete, soluble, and functional "proteinosome" structures could lead to advances in fields ranging from membrane protein production to drug delivery applications.MmsF | proteinosome | magnetite | magnetosome | in vitro precipitation M agnetic nanoparticles (MNPs) represent an area of intense research due to their diverse and pertinent applications across a range of disciplines and industries. Applications for MNPs include biomedical diagnostics and therapies (1-3), such as MRI contrast reagents, tumor hyperthermia treatments, and magnetically targeted drug delivery, as well as data storage (4) and biotechnology. However, specific magnetic and physical properties of MNPs are critical to the success of each application, with specific size and morphology (with a narrow distribution) being essential considerations. Pure magnetite MNP synthesis under ambient conditions is notoriously difficult to control, with simple precipitations often resulting in a mixture of differently sized and shaped particles with other iron oxide contaminants. This situation can be improved somewhat by using more extreme processes, such as high-temperature incubations or capping surfactants, which favor certain MNP types (5, 6). However, the use of toxic or organic reagents and extreme conditions comes with a high energy and monetary cost, and can limit the biocompatibility of the MNPs for subsequent app...

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Section: Resultsmentioning

confidence: 99%

Self-assembled MmsF proteinosomes control magnetite nanoparticle formation in vitro

Rawlings

Bramble

Walker

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The extracted lipids were dried under nitrogen and concentrated 50-fold in chloroform:methanol (1:2), and the mass spectra were generated via collision-induced dissociation electrospray mass spectrometry on a VG Quattro spectrometer at a cone voltage of 90 V. This cone voltage resulted in complete degradation of the phospholipids into the head groups and fatty acid components, and mass ranges from 100 to 500 m/z were typically collected. Fatty acids were identified based on their molecular masses (Sweetman et al 1996).…”

Section: Analysis and Extraction Of Phospholipidsmentioning

confidence: 99%

The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis

Morgan‐Kiss

Cronan

2008

Arch Microbiol

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The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacylacyl carrier protein synthase. The strain tested was a derivative (called the ΔfabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ΔfabH bypass strain that overproduced FabF and the wild type incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ΔfabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.

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“…To test this implication, we isolated phospholipids from mutant strain K165 and parental strain SC122 grown at 30°C. The lipids were analyzed by collision-induced dissociation electrospray mass spectroscopy (26) (Fig. 4).…”

Section: E Coli Cfa Synthase Is Unstable In Vivomentioning

confidence: 99%

Metabolic Instability of Escherichia coli Cyclopropane Fatty Acid Synthase Is Due to RpoH-Dependent Proteolysis

Chang¹,

Eichel

Cronan

2000

J Bacteriol

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Cyclopropane fatty acids (CFAs) are generally synthesized as bacterial cultures enter stationary phase. In Escherichia coli, the onset of CFA synthesis results from increased transcription of cfa, the gene encoding CFA synthase. However, the increased level of CFA synthase activity is transient; the activity quickly declines to the basal level. We report that the loss of CFA activity is due to proteolytic degradation dependent on expression of the heat shock regulon. CFA synthase degradation is unaffected by mutations in the lon, clpP, and groEL genes or by depletion of the intracellular ATP pools. It seems likely that CFA synthase is the target of an unidentified energy-independent heat shock regulon protease. This seems to be the first example of heat shock-dependent degradation of a normal biosynthetic enzyme.Cyclopropane fatty acids (CFAs) are very widely distributed among the bacteria and are formed by addition of a methylene group, derived from the methyl group of S-adenosyl-L-methionine (AdoMet), across the carbon-carbon double bond of unsaturated fatty acids (UFAs) (11). Methylene addition does not involve free fatty acids or intermediates of phospholipid biosynthesis, but rather, uses mature phospholipid molecules previously incorporated into and functioning within membrane bilayers. CFAs are typically produced at the onset of stationary phase in bacterial cultures, and CFA formation can thus be considered a conditional, postsynthetic modification of bacterial membrane lipid bilayers. This modification is catalyzed by a soluble enzyme, despite the fact that one of the substrates, the UFA double bond, is normally sequestered deep within the hydrophobic interior of the phospholipid bilayer. This and other properties imply topologically novel protein-lipid interactions in the biosynthesis of CFAs. No physiological role for CFA formation had been ascribed until we recently reported that Escherichia coli strains lacking CFA survive poorly when exposed to pH 3 (2).Only a small fraction of UFAs are converted to CFAs in exponentially growing cultures of E. coli and other bacteria (11). However, as cultures reach stationary phase, the UFAs of the membrane phospholipid bilayers are rapidly and almost quantitatively converted to CFA. Wang and Cronan (28) showed that the growth-phase dependence of CFA synthesis is due to activation of an RpoS-dependent cfa promoter. The cfa gene encodes CFA synthase, the 43-kDa enzyme catalyzing the modification (29), and is transcribed from two promoters, P1 and P2 (28). Promoter P1 is a standard RpoD ( 70 ) promoter and is active throughout the growth curve, whereas function of promoter P2 requires the stationary-phase sigma factor RpoS ( 38 ). The onset of stationary phase results in efficient conversion of phospholipid UFA moieties to CFAs by two mechanisms, the accumulation of RpoS gives increased cfa expression, and the decreased phospholipid synthetic rate allows CFA synthesis to catch up to phospholipid synthesis (CFA synthase must no longer cope with a continually increasi...

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Electrospray ionization mass spectrometric analysis of phospholipids of Escherichia coli

Cited by 29 publications

References 0 publications

Self-assembled MmsF proteinosomes control magnetite nanoparticle formation in vitro

Self-assembled MmsF proteinosomes control magnetite nanoparticle formation in vitro

The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis

Metabolic Instability of Escherichia coli Cyclopropane Fatty Acid Synthase Is Due to RpoH-Dependent Proteolysis

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