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Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

ABSTRACT: The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-L-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel.

SUBMITTER: Freeman JL

PROVIDER: S-EPMC1317400 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

Freeman John L JL Persans Michael W MW Nieman Ken K Salt David E DE

Applied and environmental microbiology 20051201 12

The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction ...[more]

PMID: 16332856

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Project description:Thlaspi goesingense is able to hyperaccumulate extremely high concentrations of Ni when grown in ultramafic soils. Recently it has been shown that rhizosphere bacteria may increase the heavy metal concentrations in hyperaccumulator plants significantly, whereas the role of endophytes has not been investigated yet. In this study the rhizosphere and shoot-associated (endophytic) bacteria colonizing T. goesingense were characterized in detail by using both cultivation and cultivation-independent techniques. Bacteria were identified by 16S rRNA sequence analysis, and isolates were further characterized regarding characteristics that may be relevant for a beneficial plant-microbe interaction-Ni tolerance, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore production. In the rhizosphere a high percentage of bacteria belonging to the Holophaga/Acidobacterium division and alpha-Proteobacteria were found. In addition, high-G+C gram-positive bacteria, Verrucomicrobia, and microbes of the Cytophaga/Flexibacter/Bacteroides division colonized the rhizosphere. The community structure of shoot-associated bacteria was highly different. The majority of clones affiliated with the Proteobacteria, but also bacteria belonging to the Cytophaga/Flexibacter/Bacteroides division, the Holophaga/Acidobacterium division, and the low-G+C gram-positive bacteria, were frequently found. A high number of highly related Sphingomonas 16S rRNA gene sequences were detected, which were also obtained by the cultivation of endophytes. Rhizosphere isolates belonged mainly to the genera Methylobacterium, Rhodococcus, and Okibacterium, whereas the majority of endophytes showed high levels of similarity to Methylobacterium mesophilicum. Additionally, Sphingomonas spp. were abundant. Isolates were resistant to Ni concentrations between 5 and 12 mM; however, endophytes generally tolerated higher Ni levels than rhizosphere bacteria. Almost all bacteria were able to produce siderophores. Various strains, particularly endophytes, were able to grow on ACC as the sole nitrogen source.

Project description:Although SAT (serine acetyltransferase) of Escherichia coli, which catalyses the first step in cysteine synthesis, proceeds via a random-order ternary complex reaction mechanism [Hindson and Shaw (2003) Biochemistry 42, 3113-3119], it has been suggested that the nearly identical enzyme from Salmonella typhimurium might involve an acetyl-enzyme intermediate [Leu and Cook (1994) Protein Peptide Lett. 1, 157-162]. In this study the alternative acetyl acceptor threonine and the alternative acyl donor, propionyl-CoA were used to further investigate the reaction mechanism of SAT from E. coli. Steady-state kinetic data and dead-end inhibition studies were again diagnostic of a random-order ternary complex reaction mechanism for alternative substrates. Since earlier kinetic studies with SAT from S. typhimurium suggested that cysteine competes with acetyl-CoA for binding, rather than serine with which it is isostructural, the specificity of the serine-binding pocket was assessed with three substrate mimics; beta-hydroxypropionic acid, glycine and ethanolamine. The data show that SAT interacts productively with the amino and hydroxymethyl moieties of serine, whereas the carboxyl group provides an essential contribution to binding strongly, supporting a view that cysteine will interact productively at the serine-binding site. Furthermore, since the hydroxymethyl contact region of the serine-binding site appears able to accommodate the methylene and acetyl moeties of threonine and O -acetyl-serine respectively, the site is unlikely to provide obligatory short-range contacts with the hydroxyl group of serine, a prerequisite for exclusion of cysteine. Such a proposal is supported by the results of micro-calorimetric studies which show that cysteine competes with serine for binding to SAT rather than with CoA. It follows that tight binding of cysteine at the serine-binding site near the catalytic centre may be the effector of a substantial reduction in the affinity of SAT for CoA, yielding the observed pattern of steady-state inhibition and the mechanism by which cysteine mediates effective end-product control of its synthesis.

Dataset Information

Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

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Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

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