Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation

Augustin, Johannes; Götz, Friedrich

doi:10.1016/0378-1097(90)90283-v

Cited by 140 publications

(125 citation statements)

References 18 publications

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“…aureus shuttle vector. The resulting pCL-ALDKO plasmid was electrotransformed into S. aureus RN4220 (24) and incubated at 43°C on B-mediumagar plates containing 10 g/ml erythromycin (BM-agar Erm 10 plates) for 2 days. Colonies were restreaked onto BM-agar Erm 10 plates and incubated at 43°C for 2 days.…”

Section: Methodsmentioning

confidence: 99%

Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

Kim

Lee

2012

Journal of Biological Chemistry

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Background:The biosynthetic pathway for staphyloxanthin has previously been proposed to consist of five enzymes. Results: A sixth pathway enzyme, 4,4Ј-diaponeurosporen-aldehyde dehydrogenase, was identified using a synthetic module approach. Conclusion:The complete staphyloxanthin biosynthetic pathway consists of six enzymes in Staphylococcus aureus. Significance: This is the first report demonstrating the complete staphyloxanthin pathway.The biosynthetic pathway for staphyloxanthin, a C 30 carotenoid biosynthesized by Staphylococcus aureus, has previously been proposed to consist of five enzymes (CrtO, CrtP, CrtQ, CrtM, and CrtN). Here, we report a missing sixth enzyme, 4,4-diaponeurosporen-aldehyde dehydrogenase (AldH), in the staphyloxanthin biosynthetic pathway and describe the functional expression of the complete staphyloxanthin biosynthetic pathway in Escherichia coli. When we expressed the five known pathway enzymes through artificial synthetic operons and the wild-type operon (crtOPQMN) in E. coli, carotenoid aldehyde intermediates such as 4,4-diaponeurosporen-4-al accumulated without being converted into staphyloxanthin or other intermediates. We identified an aldH gene located 670 kilobase pairs from the known staphyloxanthin gene cluster in the S. aureus genome and an aldH gene in the non-staphyloxanthin-producing Staphylococcus carnosus genome. These two putative enzymes catalyzed the missing oxidation reaction to convert 4,4-diaponeurosporen-4-al into 4,4-diaponeurosporenoic acid in E. coli. Deletion of the aldH gene in S. aureus abolished staphyloxanthin biosynthesis and caused accumulation of 4,4-diaponeurosporen-4-al, confirming the role of AldH in staphyloxanthin biosynthesis. When the complete staphyloxanthin biosynthetic pathway was expressed using an artificial synthetic operon in E. coli, staphyloxanthin-like compounds, which contained altered fatty acid acyl chains, and novel carotenoid compounds were produced, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes.

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Section: Methodsmentioning

confidence: 99%

Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

Kim

Lee

2012

Journal of Biological Chemistry

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“…Strain DU5873, a mutant of strain Newman lacking expression of protein A (strain Newman ⌬spa::Tc R ) (14) was used for the antibody inhibition studies. The shuttle plasmid pCU1 (24), which confers resistance to chloramphenicol in S. aureus and ampicillin in E. coli, was used to express the wild-type and mutant ClfA proteins in strain DU5941. Plasmids pQE30 (QIAGEN Inc.) and pGEX-KG (25) were used for recombinant protein expression.…”

Section: Methodsmentioning

confidence: 99%

Identification of Residues in the Staphylococcus aureus Fibrinogen-binding MSCRAMM Clumping Factor A (ClfA) That Are Important for Ligand Binding

Hartford¹,

Wann²,

Höök³

et al. 2001

Journal of Biological Chemistry

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“…per ml before buffer S2 was added. Transformation and cloning procedures for S. carnosus have been described (3,13). E. coli-specific recombinant DNA techniques were performed as described by Sambrook et al (32).…”

Section: Methodsmentioning

confidence: 99%

Analysis of the Staphylococcus epidermidis genes epiF, -E, and -G involved in epidermin immunity

1996

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The lantibiotic epidermin is produced by Staphylococcus epidermidis Tü3298. The known genes involved in epidermin biosynthesis and regulation are organized as operons (epiABCD and epiQP) that are encoded on the 54-kb plasmid pTü32. Here we describe the characterization of a DNA region that mediates immunity and increased epidermin production, located upstream of the structural gene epiA. The sequence of a 2.6-kb DNA fragment revealed three open reading frames, epiF, -E, and -G, which may form an operon. In the cloning host Staphylococcus carnosus, the three genes mediated an increased tolerance to epidermin, and the highest level of immunity (sevenfold) was achieved with S. carnosus carrying epiFEG and epiQ. The promoter of the first gene, epiF, responded to the activator protein EpiQ and contained a palindromic sequence similar to the EpiQ binding site of the epiA promoter, which is also activated by EpiQ. Inactivation of epiF, -E, or -G resulted in the complete loss of the immunity phenotype. An epidermin-sensitive S. epidermidis Tü3298 mutant was complemented by a DNA fragment containing all three genes. When the epiFEG genes were cloned together with plasmid pTepi14, containing the biosynthetic genes epiABCDQP, the level of epidermin production was approximately fivefold higher. The proteins EpiF, -E, and -G are similar in deduced sequence and proposed structure to the components of various ABC transporter systems. EpiF is a hydrophilic protein with conserved ATP-binding sites, while EpiE and -G have six alternating hydrophobic regions and very likely constitute the integral membrane domains. When EpiF was overproduced in S. carnosus, it was at least partially associated with the cytoplasmic membrane. A potential mechanism for how EpiFEG mediates immunity is discussed.Epidermin (1) is a well-characterized member of the lantibiotics, a group of peptide antibiotics that are distinguished by the presence of the rare amino acid lanthionine (for reviews, see references 16 and 31). The bactericidal action is mainly caused by pore formation in the cytoplasmic membrane (30). Epidermin, like all other lantibiotics found so far, is ribosomally synthesized. The structural gene for the epidermin precursor peptide, epiA, is encoded on the 54-kb plasmid pTü32 of Staphylococcus epidermidis Tü3298 (34). A 14-kb DNA fragment, sufficient for low-level epidermin production, has been subcloned in Staphylococcus carnosus TM300 (4). The nucleotide sequence revealed the presence of seven genes that are organized in three transcriptional units (33). epiA is cotranscribed with epiB, -C, and -D, which are involved in the posttranslational modification of epidermin. The flavin mononucleotide-containing enzyme EpiD, which is unique to the epidermin system, has been recently shown to catalyze the oxidative decarboxylation of the C terminus of pre-epidermin (21-23). EpiB and EpiC are proposed to catalyze the dehydration of serine and threonine residues and the formation of thioether bonds. Genes related to epiB and -C are involved in the...

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Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation

Cited by 140 publications

References 18 publications

Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

Identification of Residues in the Staphylococcus aureus Fibrinogen-binding MSCRAMM Clumping Factor A (ClfA) That Are Important for Ligand Binding

Analysis of the Staphylococcus epidermidis genes epiF, -E, and -G involved in epidermin immunity

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