Amino Acid Requirements for the Production of Enterotoxin B by Staphylococcus aureus S-6 in a Chemically Defined Medium

Miller, Richard D.; Fung, Daniel Y. C.

doi:10.1128/aem.25.5.800-806.1973

Cited by 27 publications

(22 citation statements)

References 10 publications

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“…Since superinfection of competent cells of strain 8325-4(Xll) at high multiplicities increased the frequency of transfection (20), we also tested the effect of superinfection on the transformation. The addition of phage 11 to the transformation mixture did not increase the transformation frequency ( Table 6).…”

Section: Resultsmentioning

confidence: 96%

“…To be able to isolate amino acid-requiring mutants of strain 8325, we investigated the basal amino acid requirement of this strain. Several authors (1,9,11) have reported on various salt solutions for culturing S. aureus. The salt mixture reported in Table 2 supported the best growth in media containing amino acids, glucose, and vitamins.…”

Section: Resultsmentioning

confidence: 99%

“…Centrifugation of the transformation mixture at 4 C for 10 min and suspension of the cells in TSB before plating resulted in an increased number of transformants and a broader effective concentration range of Ca2+ (Fig. 4, upper In two experiments, 11 phages at a multiplicity of infection of 1 were added to cells of strain 8325-4 thy suspended in transformation buffer with DNA from the donor strain 8325(PI2.8). Phage 11 was propagated on the recipient strain.…”

Section: Resultsmentioning

confidence: 99%

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Factors Affecting Competence for Transformation in Staphylococcus aureus

et al. 1974

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A chemically defined medium has been developed for isolation of amino acid-requiring mutants of Staphylococcus aureus strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage 011 can be transformed. Superinfection of competent cells with ,11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (ero) and the chromosomal markers trp, thy, and cyt are of the same order of magnitude, whereas the frequency for the chromosomal marker tyr is approximately one order of magnitude lower. cedure for transformation, the recipient cells were grown on TSA plates and pyrimidine-requiring mutants were grown on TSA plates with added pyrimidine. Both cultures grew at 37 C overnight and then were suspended in TSB medium to an optical density at 524 nm (OD524) of 0.100, which equals 5 x 107 colony-forming units (CFU) per milliliter. The cell suspension was then diluted 10 times in TSB medium and incubated on a rotary shaker at 37 C. Maximal competence is reached after 1.5 to 2 h of growth (somewhat dependent on the strain used; see Fig. 1 and 2). The competent cells were washed once in 0.15 M NaCl and then suspended in 0.1 M tris(hydroxymethyl)aminomethane (Tris)-maleate buffer (pH 7.0) containing 0.1 M CaCl2 at a cell density of approximately 109 CFU/ml. In the transformation experiments, 0.9 ml of this cell suspension was mixed with 0.1 ml of DNA solution to give saturating concentration of DNA, i.e., 10 ug/ml (8). After incubation at 30 C for 20 min, the cells were centrifuged and suspended in 1 ml of TSB 155 on July 31, 2020 by guest

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Factors Affecting Competence for Transformation in Staphylococcus aureus

et al. 1974

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“…We examined the growth profiles of strains in a medium that supports only respiratory growth. S. aureus is capable of utilizing glucose as well as glutamate as a source of carbon (47,48). Glutamate feeds directly into the TCA cycle at the ␣-ketoglutarate entry point, and we reasoned that glutamate would support respiratory growth almost exclusively.…”

Section: Figmentioning

confidence: 99%

The ClpCP Complex Modulates Respiratory Metabolism in Staphylococcus aureus and Is Regulated in a SrrAB-Dependent Manner

et al. 2019

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The staphylococcal respiratory regulator (SrrAB) modulates energy metabolism in Staphylococcus aureus. Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis in S. aureus is achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA ϩ family ATPase (typically, ClpC or ClpX). In the present report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis in S. aureus. Strains deficient in one or more Clp complexes were attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A ΔsrrAB strain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the ΔsrrAB strain was a result of decreased ClpC activity. Consistent with this, transcriptional activity of clpC was decreased in the ΔsrrAB mutant, and overexpression of clpC suppressed the puromycin sensitivity of the ΔsrrAB strain. We also found that ClpC positively influenced respiration and that it did so upon association with ClpP. In contrast, ClpC limited fermentative growth, while ClpP was required for optimal fermentative growth. Metabolomics studies demonstrated that intracellular metabolic profiles of the ΔclpC and ΔsrrAB mutants were distinct from those of the wild-type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis. IMPORTANCE Oxygen is used as a substrate to derive energy by the bacterial pathogen Staphylococcus aureus during infection; however, S. aureus can also grow fermentatively in the absence of oxygen. To successfully cause infection, S. aureus must tailor its metabolism to take advantage of respiratory activity. Different proteins are required for growth in the presence or absence of oxygen; therefore, when cells transition between these conditions, several proteins would be expected to become unnecessary. In this report, we show that regulated proteolysis is used to modulate energy metabolism in S. aureus. We report that the ClpCP protein complex is involved in specifically modulating aerobic respiratory growth but is dispensable for fermentative growth.

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“…Attempts have been made to determine the minimum requirements for enterotoxin production in chemically defined media , Wu & Bergdoll 1971Miller & Fung 1973). Whilst toxin production occurred in defined media containing essential salts, vitamins, and an energy source such as glucose, glutamate or amino acids, in general it was never as high as could be achieved in complex media.…”

Section: Culture Mediamentioning

confidence: 99%