Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Peschel A

PROVIDER: S-EPMC177688 | biostudies-other | 1996 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

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

Peschel A A Götz F F

Journal of bacteriology 19960101 2

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 ...[more]

PMID: 8550476

Similar Datasets

Project description:Group A Streptococcus (GAS) is one of the world’s most successful pathogens, causing a multitude of common infections such as pharyngitis, cellulitis, and impetigo. It is also responsible for more severe diseases such as rheumatic fever, necrotizing fasciitis, and toxic shock syndrome. Group A Streptococcus produces a powerful peptide toxin known as streptolysin S (SLS). Although recent advances have begun to uncover the structure of SLS, many questions remain as to its route of synthesis, export and function. A fundamental yet unanswered question about SLS is the mechanism employed by GAS to resist the effects of its own toxin. To address this question, we developed a unique microarray-based approach aimed at identifying bacterial genes involved in SLS immunity. We measured changes in gene expression in a non-SLS producing GAS strain (∆SLS) after exposure to active SLS, as well as to an inactive SLS isoform. Initial exposure of a non-SLS producing GAS to the native SLS toxin resulted in significant upregulation of several gene candidates. Proteins encoded by these gene candidates were produced and tested for their ability to neutralize SLS toxin activity in vitro. The protein encoded by the gene Spy_0787 decreased the cytolytic activity of wt SLS by 50%. Bacterial immunity is still a relatively unknown and unexplained phenomena. Insights into how toxin-producing microorganisms achieve resistance against the effects of their own toxin could uncover important therapeutic targets to neutralize virulence factors such as SLS, as well as other related peptide toxins. The microarray technique described here can be leveraged to other microbial systems to understand how microorganisms in general react to antibacterial and cytotoxic compounds for which the mechanism of action is unknown. Supernatants containing wt and S39A forms of SLS secreted by GAS strains were collected from 50 ml of overnight cultures of each strain at 37 degrees C in Todd Hewett broth supplemented with 10 mg/ml of bovine serum albumin fraction V (Sigma chemicals) in order to stabilize the toxin. The supernatant was then isolated and subjected to filter sterilization. For SLS exposure conditions, bacterial pellets containing GAS ∆SagA mutants were incubated with 1. supernatants containing wtSLS; 2. supernatants containing SLS S39A isoform; or 3. sterile TH media with 10mg/ml of BSA. The bacteria pellets were quickly resuspended after which the bacterial pellets were recollected for RNA isolation. A total of three exposure timepoints were used: 0h (immediately after exposure to SLS-containing supernatants), 3h post-exposure, and 6h post-exposure. The pellets were frozen at -80C until used for RNA extraction. Total RNA extraction and purification from the bacterial pellets were performed using the RNeasy isolation kit per manufacturer's instructions (Qiagen). RNA samples were processed following standard Roche NimbleGen gene expression analysis protocols. Double-stranded cDNA was synthesized from 10 μg of total RNA using random hexamer primers with the SuperScript cDNA synthesis kit (Invitrogen). One μg of cDNA was labeled with Cy3-random nonamers (Roche NimbleGen) for microarray hybridization.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets