Cloning and Characterization of the Phosphatidylserine Synthase Gene of
            <i>Agrobacterium</i>
            sp. Strain ATCC 31749 and Effect of Its Inactivation on Production of High-Molecular-Mass (1→3)-β-
            <scp>d</scp>
            -Glucan (Curdlan)

Karnezis, Tara; Fisher, Helen C.; Neumann, Gregory M.; Stone, Bruce; Stanisich, Vilma A.

doi:10.1128/jb.184.15.4114-4123.2002

Cited by 45 publications

(42 citation statements)

References 63 publications

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“…(29) and Agrobacterium spp. (16), the lack of PE does not affect the growth of Brucella in rich complex medium. However, in minimal defined medium, the B. abortus pssA mutant showed a strong dependence on choline for growth, indicating that PC synthesis is essential in a PE-deficient background.…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of Brucella abortus

et al. 2008

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The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.

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Section: Discussionmentioning

confidence: 99%

Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of Brucella abortus

et al. 2008

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“…5A), we speculate that PmtA requires a defined phospholipid environment, specifically the presence of PG, for full enzymatic activity. A. tumefaciens membranes are known to possess PG (23,34). Since PG stimulates PE, MMPE, or DMPE methylation, it seems likely that PG binds to PmtA at a site different from the substrate binding site.…”

Section: Discussionmentioning

confidence: 99%

“…It was discovered more than 40 years ago that the plant pathogen A. tumefaciens produces PC by two alternative pathways, the methylation pathway (20) and the phosphatidylcholine synthase pathway (23,29,34). PE methylation activity was demonstrated in partially purified A. tumefaciens extracts (20) but has not been studied since this seminal finding.…”

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confidence: 99%

In Vitro Characterization of the Enzyme Properties of the Phospholipid N -Methyltransferase PmtA from Agrobacterium tumefaciens

Aktas

Narberhaus

2009

J Bacteriol

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Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

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“…Electrospray ionization mass spectrometry was performed as described by Karnezis et al (32). To identify potential FP-binding proteins, peptide masses obtained from the mass spectrometric analyses were used in the search engine ProFound, to match data with the NCBI nonredundant data base entries (129.85.19.192/profound_bin/WebProfound.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Heme-interacting Proteins in the Malaria Parasite, Plasmodium falciparum

Campanale

Nickel²,

Daubenberger

et al. 2003

Journal of Biological Chemistry

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The degradation of hemoglobin by the malaria parasite, Plasmodium falciparum, produces free ferriprotoporphyrin IX (FP) as a toxic by-product. In the presence of FP-binding drugs such as chloroquine, FP detoxification is inhibited, and the build-up of free FP is thought to be a key mechanism in parasite killing. In an effort to identify parasite proteins that might interact preferentially with FP, we have used a mass spectrometry approach. Proteins that bind to FP immobilized on agarose include P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), P. falciparum glutathione reductase (PfGR), and P. falciparum protein disulfide isomerase. To examine the potential consequences of FP binding, we have examined the ability of FP to inhibit the activities of GAPDH and GR from P. falciparum and other sources. FP inhibits the enzymic activity of Pf-GAPDH with a K i value of 0.2 M, whereas red blood cell GAPDH is much less sensitive. By contrast, PfGR is more resistant to FP inhibition (K i > 25 M) than its human counterpart. We also examined the ability of FP to inhibit the activities of the additional antioxidant enzymes, P. falciparum thioredoxin reductase, which exhibits a K i value of 1 M, and P. falciparum glutaredoxin, which shows more moderate sensitivity to FP. The exquisite sensitivity of PfGAPDH to FP may indicate that the glycolytic pathway of the parasite is particularly susceptible to modulation by FP stress. Inhibition of this pathway may drive flux through the pentose phosphate pathway ensuring sufficient production of reducing equivalents to counteract the oxidative stress induced by FP build-up.

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Cloning and Characterization of the Phosphatidylserine Synthase Gene of Agrobacterium sp. Strain ATCC 31749 and Effect of Its Inactivation on Production of High-Molecular-Mass (1→3)-β- d -Glucan (Curdlan)

Cited by 45 publications

References 63 publications

Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of Brucella abortus

Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of Brucella abortus

In Vitro Characterization of the Enzyme Properties of the Phospholipid N -Methyltransferase PmtA from Agrobacterium tumefaciens

Identification and Characterization of Heme-interacting Proteins in the Malaria Parasite, Plasmodium falciparum

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