Staphylococcus carnosus: from starter culture to protein engineering platform

Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and “food grade” species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.

     

The Role of Solution Conditions in the Bacteriophage PP7 Capsid Charge Regulation

We investigate and quantify the effects of pH and salt concentration on the charge regulation of the bacteriophage PP7 capsid. These effects are found to be extremely important and substantial, introducing qualitative changes in the charge state of the capsid such as a transition from net-positive to net-negative charge depending on the solution pH. The overall charge of the virus capsid arises as a consequence of a complicated balance with the chemical dissociation equilibrium of the amino acids and the electrostatic interaction between them, and the translational entropy of the mobile solution ions, i.e., counterion release. We show that to properly describe and predict the charging equilibrium of viral capsids in general, one needs to include molecular details as exemplified by the acid-base equilibrium of the detailed distribution of amino acids in the proteinaceous capsid shell.     

The repair of DNA damages induced in normal human skin fibroblasts exposed to simulated sunlight

The induction and repair of DNA damages produced by exposure of normal human skin fibroblasts to the simulated sunlight produced by a solar simulator were examined. The photoproducts measured were pyrimidine dimers, E. coli endonuclease III-sensitive sites, 6-4 photoproducts, Dewar isomers, DNA-protein crosslinks, and DNA single-strand breaks. The results of these experiments serve to form a basis for the quantitation of damages induced by exposure to sunlight.     

Orthognathic surgery in cleft patients treated by early bone grafting

For the past 25 years at Children's Memorial Hospital in Chicago a protocol has been followed for complete clefts that involves placement of an infant maxillary orthopedic appliance prior to lip closure, surgical closure of the lip, autogenous split-rib grafts to the alveolus to stabilize maxillary segments, and palatal closure, generally within the first year of life. The oldest 36 patients whose skeletal growth was for all practical purposes finished have been followed to determine the need for and type of orthognathic surgery. Of the total sample, 8 patients (22.2 percent) required some type of sagittal orthognathic surgery (1 patient in this group also required vertical maxillary alignment) and 2 patients required maxillary augmentation only in the form of an onlay graft. This report may serve as a baseline for others who wish to report on the incidence and type of orthognathic surgery in their cleft palate centers.     

The case for early bone grafting in cleft lip and palate: a second report

In 1982, the first long-term study of our early bone-grafting and infant maxillary orthopedic approach to newborn complete clefts of the lip, alveolus, and palate was published. The protocol and sequence of procedures were shown on the first 16 consecutively treated orthodontic patients, with a mean age of 14 years. Cephalometric analysis evaluated anteroposterior and vertical facial growth. This report follows the next 37 consecutively treated individuals in a similar manner and includes not only 20 complete unilateral clefts, but also 17 complete bilateral clefts. Results, when evaluated alone and in comparison with the original series, show once again that there are no adverse growth restraints and that early primary bone grafting in our protocol leads to teeth in better overall occlusion than if it had not been undertaken.     

Characterization of DNA repair in a mutant cell line derived from ICR 2A frog cells that is hypersensitive to non-dimer DNA damages induced by solar ultraviolet radiation

The level of excision repair and the inhibition and recovery of semiconservative DNA synthesis were examined following the induction of non-dimer DNA damages by solar ultraviolet radiation in a mutant cell line, DRP 36, derived from ICR 2A frog cells that is hypersensitive to these lesions. A relatively pure population of non-dimer photoproducts was produced by exposure of cells to the Mylar-filtered solar UV wavelengths produced by a fluorescent sunlamp followed by treatment with photoreactivating light (PRL) which removes most of the small yield of dimers induced by the irradiation. Using a modification of the bromodeoxyuridine (BrdUrd) photolysis assay, that enhances the sensitivity of this assay, it was found that DRP 36 cells perform a significantly lower level of excision repair following the induction of non-dimer DNA damages compared with the ICR 2A cells. In contrast, the level of excision repair of 254-nm-induced dimers was similar in the two cell lines. In addition, the induction of non-dimer damages caused a greater inhibition of DNA synthesis that persisted for a longer period of time in the mutant compared with the parental cells. Hence, these results indicate that the DRP 36 cells are deficient in the repair of at least one type of solar UV-induced non-dimer lesion.