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Crystallization of the fimbrial protein FimP from Actinomyces oris and of a triple Ile-to-Met mutant engineered to facilitate selenomethionine labelling.

ABSTRACT: Actinomyces oris is an oral bacterium important for the development of dental plaque. It expresses two forms of fimbriae: type 1 and type 2. FimP, which is the fimbrial protein that is polymerized into the stalk of the type 1 fimbriae, was cloned, overexpressed and crystallized. X-ray data were collected and processed to 2.2 Å resolution. The crystals belonged to space group P2(1)2(1)2, with one molecule in the asymmetric unit. To facilitate structure determination using single anomalous dispersion, three methionines were introduced by site-directed mutagenesis. Crystals of selenomethionine-labelled protein were obtained by streak-seeding and diffracted to 2.0 Å resolution.

SUBMITTER: Persson K

PROVIDER: S-EPMC3212363 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Crystallization of the fimbrial protein FimP from Actinomyces oris and of a triple Ile-to-Met mutant engineered to facilitate selenomethionine labelling.

Persson Karina K

Acta crystallographica. Section F, Structural biology and crystallization communications 20110924 Pt 10

Actinomyces oris is an oral bacterium important for the development of dental plaque. It expresses two forms of fimbriae: type 1 and type 2. FimP, which is the fimbrial protein that is polymerized into the stalk of the type 1 fimbriae, was cloned, overexpressed and crystallized. X-ray data were collected and processed to 2.2 Å resolution. The crystals belonged to space group P2(1)2(1)2, with one molecule in the asymmetric unit. To facilitate structure determination using single anomalous dispers ...[more]

PMID: 22102028

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Project description:BACKGROUND:Actinomyces oris is a Gram-positive bacterium that has been associated with healthy and diseased sites in the human oral cavity. Most pathogenic bacteria require iron to survive, and in order to acquire iron in the relatively iron-scarce oral cavity A. oris has been shown to produce iron-binding molecules known as siderophores. The genes encoding these siderophores and transporters are thought to be regulated by the amount of iron in the growth medium and by the metal-dependent repressor, AmdR, which we showed previously binds to the promoter of proposed iron-regulated genes. OBJECTIVE:The purpose of this study was to characterize siderophore and associated iron transport systems in A. oris. DESIGN:We examined gene expression of the putative iron transport genes fetA and sidD in response to low- and high-iron environments. One of these genes, sidD, encoding a putative Fe ABC transporter protein, was insertionally inactivated and was examined for causing growth defects. To gain a further understanding of the role of iron metabolism in oral diseases, clinical isolates of Actinomyces spp. were examined for the presence of the gene encoding AmdR, a proposed global regulator of iron-dependent gene expression in A. oris. RESULTS:When A. oris was grown under iron-limiting conditions, the genes encoding iron/siderophore transporters fetA and sidD showed increased expression. One of these genes (sidD) was mutated, and the sidD::Km strain exhibited a 50% reduction in growth in late log and stationary phase cells in media that contained iron. This growth defect was restored when the sidD gene was provided in a complemented strain. We were able to isolate the AmdR-encoding gene in seven clinical isolates of Actinomyces. When these protein sequences were aligned to the laboratory strain, there was a high degree of sequence similarity. CONCLUSIONS:The growth of the sidD::Km mutant in iron-replete medium mirrored the growth of the wild-type strain grown in iron-limiting medium, suggesting that the sidD::Km mutant was compromised in iron uptake. The known iron regulator AmdR is well conserved in clinical isolates of A. oris. This work provides additional insight into iron metabolism in this important oral microbe.

Dataset Information

Crystallization of the fimbrial protein FimP from Actinomyces oris and of a triple Ile-to-Met mutant engineered to facilitate selenomethionine labelling.

Publications

Crystallization of the fimbrial protein FimP from Actinomyces oris and of a triple Ile-to-Met mutant engineered to facilitate selenomethionine labelling.

Similar Datasets

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