Chapter 10 Using Phosphopantetheinyl Transferases for Enzyme Posttranslational Activation, Site Specific Protein Labeling and Identification of Natural Product Biosynthetic Gene Clusters from Bacterial Genomes

Sünbül, Murat; Zhang, Keya; Yin, Jun

doi:10.1016/s0076-6879(09)04810-1

Cited by 22 publications

(20 citation statements)

References 67 publications

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“…Chemokines were expressed and purified as described previously [18], [21]. The Sfp enzyme was a gift from Professor Jun Yin (University of Chicago) and was expressed and purified as described previously [22]. All fluorescent probes used in this study (Table 1) were purchased from Invitrogen, with the exception of Cy3B (GE Healthcare) and NPM (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…All fluorescent probes used in this study (Table 1) were purchased from Invitrogen, with the exception of Cy3B (GE Healthcare) and NPM (Sigma). The coenzyme A (CoA)-fluorophore conjugates were prepared as described previously [22], and lyophilized following purification by C18 reversed-phase HPLC. Prior to the labeling reaction, the stock concentration of the dye conjugate was determined by the maximum UV/VIS absorbance, using the extinction coefficients published by the dye manufacturers.…”

Section: Methodsmentioning

confidence: 99%

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A General Method for Site Specific Fluorescent Labeling of Recombinant Chemokines

et al. 2014

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Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11–12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4°C, and co-internalization with their receptors at 37°C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A General Method for Site Specific Fluorescent Labeling of Recombinant Chemokines

et al. 2014

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“…The biosynthetic potential of NRPS/PKS systems in bacterial genomes can also be estimated by the number and diversity of several small marker proteins, such as MbtH homologs (60), or the number of 4=-phosphopantetheinyl transferases (61). For a total of 18 biosynthetic gene clusters embodying NRPS and type I PKS domains, only one MbtH homolog could be identified (CMC5_078140), and two genes encoding 4=-phosphopantetheinyl transferases (CMC5_018000 and CMC5_023730) are found in the 11.4-Mb chromosome of C. crocatus.…”

Section: Figmentioning

confidence: 99%

Genome Analysis of the Fruiting Body-Forming Myxobacterium Chondromyces crocatus Reveals High Potential for Natural Product Biosynthesis

Zaburannyi

Bunk

Maier

et al. 2016

Appl Environ Microbiol

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Here, we report the complete genome sequence of the type strain of the myxobacterial genus Chondromyces, Chondromyces crocatus Cm c5. It presents one of the largest prokaryotic genomes featuring a single circular chromosome and no plasmids. Analysis revealed an enlarged set of tRNA genes, along with reduced pressure on preferred codon usage compared to that of other bacterial genomes. The large coding capacity and the plethora of encoded secondary metabolite biosynthetic gene clusters are in line with the capability of Cm c5 to produce an arsenal of antibacterial, antifungal, and cytotoxic compounds. Known pathways of the ajudazol, chondramide, chondrochloren, crocacin, crocapeptin, and thuggacin compound families are complemented by many more natural compound biosynthetic gene clusters in the chromosome. Whole-genome comparison of the fruiting-bodyforming type strain (Cm c5, DSM 14714) to an accustomed laboratory strain which has lost this ability (nonfruiting phenotype, Cm c5 fr؊) revealed genetic changes in three loci. In addition to the low synteny found with the closest sequenced representative of the same family, Sorangium cellulosum, extensive genetic information duplication and broad application of eukaryotic-type signal transduction systems are hallmarks of this 11.3-Mbp prokaryotic genome. Myxobacteria represent a branch of Deltaproteobacteria with exceptionally large and GC-rich genomes. Usually isolated from soil, these bacteria are best known for a complex development cycle and as prolific producers of secondary metabolites exhibiting a broad variety of biological activities (1). The chemical diversity of the produced compounds has motivated scientists to study more myxobacterial strains, with an emphasis on phylogenetic variety (2, 3, 4). Several genomes of secondary metaboliteproducing myxobacteria have been completely sequenced (5, 6). They exhibit genome sizes ranging from 9.2 to 14.7 Mbp, including the currently largest known bacterial genome of Sorangium cellulosum So0157-2 (7). The genomic diversity of myxobacteria is vast, and even strains of the same species may undergo genome reduction or expansion at an order of millions of base pairs (6, 7). Such diversity coincides with deviations in the genomic content of secondary metabolite biosynthetic gene clusters of known genomes, ranging from a prediction of 19 such clusters in Sandaracinus amylolyticus DSM 53668 to an estimated 38 clusters in Sorangium cellulosum So0157-2. Therefore, to better understand the biosynthetic capabilities of the myxobacteria and the relative frequency of secondary metabolites encoded in their genomes, more representatives are required to be sequenced. We here report our analysis of the genome sequence of Chondromyces crocatus Cm c5, the first representative of the genus Chondromyces belonging to the family of the Sorangiineae. MATERIALS AND METHODSGenome sequencing. The genome sequence of Chondromyces crocatus Cm c5 was determined through a combination of next-and thirdgeneration technologies available at the Helmho...

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“…It proved that mutation of zmsO will not affect the transcriptional levels of polyketide synthase ZmsA (Zhou et al 2011) and non-ribosomal peptide synthase ZmsK (Cheng et al 2013) in D. zeae. Analysis of the amino acid sequence of ZmsO showed that it is a 4′-phosphopantetheinyl transferase, which usually transform 4′-phosphopantetheine moiety from CoA to apo-CPs to form holo-CPs (Beld et al 2014;Sunbul et al 2009). Previous studies have classified the 4′-phosphopantetheinyl transferases into three types (Beld et al 2014) (Fig.…”

Section: Ec1mentioning

confidence: 99%

“…This modification is carried out by transformation of 4′-phosphopantetheine moiety from coenzyme A (CoA) to apo-carrier proteins (CPs) to form holo-carrier proteins by phosphopantetheinyl transferase (PPTase) (Beld et al 2014;Sunbul et al 2009). According to the substrate specificity, PPTases were classified into three subtypes.…”

Section: Introductionmentioning

confidence: 99%

A Sfp-type phosphopantetheinyl transferase ZmsO is essential for zeamines production and the virulence of Dickeya zeae

et al. 2016

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Zeamines are family of potent antibiotics and virulence determinants produced by the rice foot rot bacterial pathogen Dickeya zeae. So zeamines are important for the pathogenesis of D. zeae and development of new strategies against this devastating disease. In this study, we show that production of zeamines is positively modulated by ZmsO, which is a conserved Sfp-type phosphopantetheinyl thransferase (PPTase) associated with post-translational activation of fatty acid synthases and polyketide synthases. Deletion of zmsO significantly abolished zeamines production and attenuated the virulence of D. zeae without affect the growth rate. The zmsO gene is located at the upstream of zmsA and zmsK in the same gene cluster. Consistent with its role in post-translational modification, deletion of zmsO did not affect the transcriptional expression of zmsA and zmsK. In trans expression of the pcpS gene from Pseudomonas aeruginosa, which encodes a Sfp-type PPTase, in the zmsO deletion mutant could also fully restore the zeamines production and bacterial virulence, establishing the important role of Sfp-type PPTase in the zeamines biosynthesis and pathogenicity of D. zeae.

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Chapter 10 Using Phosphopantetheinyl Transferases for Enzyme Posttranslational Activation, Site Specific Protein Labeling and Identification of Natural Product Biosynthetic Gene Clusters from Bacterial Genomes

Cited by 22 publications

References 67 publications

A General Method for Site Specific Fluorescent Labeling of Recombinant Chemokines

A General Method for Site Specific Fluorescent Labeling of Recombinant Chemokines

Genome Analysis of the Fruiting Body-Forming Myxobacterium Chondromyces crocatus Reveals High Potential for Natural Product Biosynthesis

A Sfp-type phosphopantetheinyl transferase ZmsO is essential for zeamines production and the virulence of Dickeya zeae

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