Protoplast transformation of Staphylococcus carnosus by plasmid DNA

Götz, Friedrich; Kreutz, Birgit; Schleifer, Karl Heinz

doi:10.1007/bf00337828

Cited by 57 publications

(23 citation statements)

References 17 publications

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“…DNA manipulations, plasmid DNA isolation, and transformation of E. coli were performed by standard procedures. S. carnosus was transformed with plasmid DNA by protoplast transformation (11,12), and S. epidermidis was transformed by electroporation (26). For DNA preparations from S. epidermidis, the cell wall was digested with 10 U lysostaphin/ml (Ambicin L, recombinant; Applied Micro Inc.).…”

Section: Methodsmentioning

confidence: 99%

Augmented Expression of Polysaccharide Intercellular Adhesin in a DefinedStaphylococcus epidermidisMutant with the Small-Colony-Variant Phenotype

et al. 2007

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While coagulase-negative staphylococci (CoNS), with their ability to form a thick, multilayered biofilm on foreign bodies, have been identified as the major cause of implant-associated infections, no data are available about biofilm formation by staphylococcal small-colony variants (SCVs). In the past years, a number of device-associated infections due to staphylococcal SCVs were described, among them, several pacemaker infections due to SCVs of CoNS auxotrophic to hemin. To test the characteristics of SCVs of CoNS, in particular, to study the ability of SCVs to form a biofilm on foreign bodies, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in Staphylococcus epidermidis. In fact, this mutant displayed a stable SCV phenotype with tiny colonies showing strong adhesion to the agar surface. When the incubation time was extended to 48 h or a higher inoculum concentration was used, the mutant produced biofilm amounts on polystyrene similar to those produced by the parent strain. When grown under planktonic conditions, the mutant formed markedly larger cell clusters than the parental strain which were completely disintegrated by the specific ␤-1,6-hexosaminidase dispersin B but were resistant to trypsin treatment. In a dot blot assay, the mutant expressed larger amounts of polysaccharide intercellular adhesin (PIA) than the parent strain. In conclusion, interrupting a hemin biosynthetic gene in S. epidermidis resulted in an SCV phenotype. Markedly larger cell clusters and the ability of the hemB mutant to form a biofilm are related to the augmented expression of PIA.

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Section: Methodsmentioning

confidence: 99%

Augmented Expression of Polysaccharide Intercellular Adhesin in a DefinedStaphylococcus epidermidisMutant with the Small-Colony-Variant Phenotype

et al. 2007

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“…Plasmid DNA from E. coli and staphylococci were purified with a Genelute plasmid miniprep kit (Sigma) according to the manufacturer's protocol, except that the staphylococcal bacterial cells were lysed by lysostaphin (Sigma; 12.5 g/ml) at 37°C for 1 h before plasmid purification. Plasmids were introduced into the staphylococci by electroporation or by protoplast transformation with previously described methods (11,19,20). Restriction enzymes were purchased from Roche and used according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

SarA Is an Essential Positive Regulator of Staphylococcus epidermidis Biofilm Development

et al. 2005

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Staphylococcus epidermidis biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA)--poly-N-acetylglucosamine polysaccharide (PNAG) by the products of the icaADBC operon. Recent evidence indicates that SarA, a central regulatory element that controls the production of Staphylococcus aureus virulence factors, is essential for the synthesis of PIA/PNAG and the ensuing biofilm development in this species. Based on the presence of a sarA homolog, we hypothesized that SarA could also be involved in the regulation of the biofilm formation process in S. epidermidis. To investigate this, we constructed nonpolar sarA deletions in two genetically unrelated S. epidermidis clinical strains, O-47 and CH845. The SarA mutants were completely defective in biofilm formation, both in the steady-state conditions of a microtiter dish assay and in the flow conditions of microfermentors. Reverse transcription-PCR experiments showed that the mutation in the sarA gene resulted in downregulation of the icaADBC operon transcription in an IcaR-independent manner. Purified SarA protein showed high-affinity binding to the icaA promoter region by electrophoretic mobility shift assays. Consequently, mutation in sarA provoked a significant decrease in the amount of PIA/PNAG on the cell surface. Furthermore, heterologous complementation of S. aureus sarA mutants with the sarA gene of S. epidermidis completely restored biofilm formation. In summary, SarA appeared to be a positive regulator of transcription of the ica locus, and in its absence, PIA/PNAG production and biofilm formation were diminished. Additionally, we present experimental evidence showing that SarA may be an important regulatory element that controls S. epidermidis virulence factors other than biofilm formation.Chronic nosocomial infections by biofilm-forming Staphylococcus epidermidis have become more prevalent in recent years with the increased use of prosthetic medical implants. Biofilm formation by S. epidermidis frequently compromises the effectiveness of implanted medical devices by giving rise to persistent and relapsing infections, which are more resistant to the host immune response and antimicrobial chemotherapy (for a review, see reference 18). The formation of S. epidermidis biofilms is proposed to occur in a two-step manner, in which a cellular accumulation process to form the mature biofilm follows rapid initial attachment to an inert synthetic surface (22). Critical to S. epidermidis biofilm formation is the production of a poly-N-acetylglucosamine polysaccharide (PNAG)-polysaccharide intercellular adhesin (PIA) (33, 34). The intercellular adhesin (icaADBC) locus, originally described in S. epidermidis (22,23) and later found in Staphylococcus aureus (9), contains the genes involved in PIA/PNAG production. The significance of PIA/PNAG as a virulence factor was demonstrated in a central venous catheter infection model of a rat and in a subcutaneous foreign-body infection model in mice (43,44). In addition, the ica operon...

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“…Plasmid pCXI9 is a derivative of the xylose-inducible expression vector pCXI5 (36). The ligation mixture was transformed into S. carnosus protoplasts (12). A representative plasmid containing eap as an insert was designated pCXEap, and accordingly, the transformed S. carnosus strain was designated TM300(pCXEap).…”

Section: Methodsmentioning

confidence: 99%

Insertional Inactivation of eap in Staphylococcus aureus Strain Newman Confers Reduced Staphylococcal Binding to Fibroblasts

et al. 2002

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In contrast, the mutant adhered to a significantly lesser extent to cultured fibroblasts (P < 0.001) than did the wild type, while adherence was restorable upon complementation. Furthermore, adherence to both epithelial cells (P < 0.05) and fibroblasts (not significant) could be blocked with antibodies against Eap, whereas preimmune serum was not active. In conclusion, Eap may contribute to pathogenicity by promoting adhesion of whole staphylococcal cells to complex eukaryotic substrates.Staphylococcus aureus continues to be a major cause of human disease, accounting for superficial skin infections as well as for serious invasive infections, such as endocarditis, osteomyelitis, and septic shock (22). Adherence of S. aureus to components of host tissues is an important first step in colonization and subsequent infection (10, 35) and is mediated by specific interactions between adhesins on the bacterial cell surface and host cell receptors (6). In addition to the wellcharacterized bacterial surface-located proteins (28) conferring adhesion to various extracellular matrix proteins (6) and invasion of eukaryotic cells (1, 31, 32), other adhesive S. aureus proteins are secreted. Three of these molecules, i.e., coagulase (29), the extracellular fibrinogen (Fg)-binding protein Efb (26), and the extracellular adherence protein Eap (24), have been shown to bind Fg. Eap of S. aureus Newman has been cloned and sequenced (14) and has previously been shown to bind to additional plasma proteins, including fibronectin (Fn) and prothrombin (Pt) (24). Eap can form oligomers, and by rebinding to the staphylococcal cell surface, it mediates bacterial agglutination. It also enhances binding to epithelial cells and fibroblasts by its dual affinity for eukaryotic components and the S. aureus surface (24). While these observations have been made with purified Eap, further precision in describing the role of Eap, as well as of the related molecule Map (for major histocompatibility complex class II analogous protein [19]), in intact S. aureus cells has been hampered by the lack of availability of defined Eap-negative mutants. Thus, we have constructed an eap-deficient mutant by allelic replacement, and here we report the genotypic and phenotypic characteristics of the mutant compared to the parent strain. MATERIALS AND METHODSBacterial strains and media. S. aureus Newman (kindly provided by T. Foster, Dublin, Ireland) was used to generate the ⌬eap mutant. Recombinant plasmids cloned in Escherichia coli were passaged in a restriction-negative S. aureus strain, SA113 (16), before electroporation to S. aureus Newman. Staphylococcus carnosus TM300 (11) was used as an intermediate host in the construction of a complemented strain. The following strains of E. coli were used as cloning hosts: E. coli DH5␣, E. coli TG1, and E. coli SCS 110 (Stratagene, La Jolla, Calif.).For cultivation of S. aureus, tryptic soy broth and agar (Difco, Detroit, Mich.), brain heart broth and agar (Merck, Darmstadt, Germany), Mueller-Hinton broth and agar (Mast, Me...

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Protoplast transformation of Staphylococcus carnosus by plasmid DNA

Cited by 57 publications

References 17 publications

Augmented Expression of Polysaccharide Intercellular Adhesin in a DefinedStaphylococcus epidermidisMutant with the Small-Colony-Variant Phenotype

Augmented Expression of Polysaccharide Intercellular Adhesin in a DefinedStaphylococcus epidermidisMutant with the Small-Colony-Variant Phenotype

SarA Is an Essential Positive Regulator of Staphylococcus epidermidis Biofilm Development

Insertional Inactivation of eap in Staphylococcus aureus Strain Newman Confers Reduced Staphylococcal Binding to Fibroblasts

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