Three subspecies of Staphylococcus sciuri, S. sciuri subsp. sciuri Moos, Schleifer, and Smith 1976, 23& emend. Moos et al. 1996, S. sciuri subsp. curnaticus subsp. nov., and S. sciuri subsp. rodentium subsp. nov., are described on the basis of their ribotype patterns, DNA-DNA liquid hybridization data, and phenotypic characteristics. Normalized ribotyping subdivided the S. sciuri patterns into three blocks of patterns, each corresponding to a subspecies. Each subspecies formed a separate, well-defined DNA similarity group when DNA-DNA hybridizations were conducted under stringent (7OOC) reassociation conditions. S. sciuri subsp. sciuri could be distinguished from the other subspecies on the basis of its ability to produce acid from D-cellobiose, alkaline phosphatase activity, and inability to produce either clumping factor or protein A. S. sciuri subsp. curnaticus could be distinguished by its ability to produce acid aerobically from D-xylose and maltose, inability to produce acid from D-melezitose, and smaller colony size on P agar. S. sciuri subsp. rodentium could be distinguished by its positive reaction in the latex agglutination test for clumping factor and/or protein A and generally higher frequencies and levels of oxacillin and methicillin resistance. All 40 strains of S. sciuri tested (including representatives of all three subspecies) hybridized with the mecA gene probe. All strains of S. sciuri subsp. sciuri, 79% of the strains of S. sciuri subsp. carnuticus and 89% of the strains of S. sciuri subsp. rodentium exhibited extracellular, staphylolytic enzyme activity. This activity was associated with an enzyme(s) that immunoblotted with a lysostaphin-specific monoclonal antibody; however, only three strains hybridized with a lysostaphin (end) gene probe. The type strain of S. sciuri subsp. curnaticus is DD 791 (= ATCC 700058), and the type strain of S. sciuri subsp. rodentium is DD 4761 (= ATCC 700061).Staphylococcus sciuri is generally considered one of the most primitive Staphylococcus species; it is widely distributed in nature, is capable of growth on inorganic nitrogen salts as the sole source of nitrogen, and exhibits a wide range of biochemical activities (31-33). In the past 20 years since its original description (33), numerous laboratories have reported frequent isolation of this species from foods, farm animals, rodents, marsupials, marine mammals, and birds and occasional isolation from humans and their pets (1,2, 17,28,31,35,48,52,57,58).Most resident populations of S. sciuri are members of the normal cutaneous microflora of lower mammals. This species is rarely associated with infections. A preliminary investigation of a large collection of S. sciuri strains showed that a ribotyping method based on an analysis of genomic EcoRI fragments containing portions of the rRNA operons could be used to distinguish two major subgroups and additional strain variation within the species (10).In this study, we describe three subpopulations of S. sciuri which can be distinguished on the basis of their rib...

Staphylococcus simulans biovar staphylolyticus produces an extracellular glycylglycine endopeptidase (lysostaphin) that lyses other staphylococci by hydrolyzing the cross bridges in their cell wall peptidoglycans. The genes for endopeptidase (end) and endopeptidase resistance (epr) reside on plasmid pACK1. An 8.4-kb fragment containing end was cloned into shuttle vector pLI50 and was then introduced into Staphylococcus aureus RN4220. The recombinant S. aureus cells produced endopeptidase and were resistant to lysis by the enzyme, which indicated that the cloned fragment also contained epr. Treatments to remove accessory wall polymers (proteins, teichoic acids, and lipoteichoic acids) did not change the endopeptidase sensitivity of walls from strains of S. simulans biovar staphylolyticus or of S. aureus with and without epr. Immunological analyses of various wall fractions showed that there were epitopes associated with endopeptidase resistance and that these epitopes were found only on the peptidoglycans of epr ؉ strains of both species. Treatment of purified peptidoglycans with endopeptidase confirmed that resistance or susceptibility of both species was a property of the peptidoglycan itself. A comparison of the chemical compositions of these peptidoglycans revealed that cross bridges in the epr ؉ cells contained more serine and fewer glycine residues than those of cells without epr. The presence of the 8.4-kb fragment from pACK1 also increased the susceptibility of both species to methicillin.

Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a D-alanyl-L-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive-and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.Streptococcal peptidoglycans (16) have a repeating backbone of the amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Connected to the lactyl group of MurNAc is a tetrapeptide (stem peptide) composed of L-alanine, D-isoglutamine (or D-isoglutamate), L-lysine, and D-alanine. The stem peptides are connected by peptide cross bridges that typically contain two or three L-alanine residues ( Fig. 1). During peptidoglycan synthesis, the stem peptides contain an additional C-terminal D-alanine ( Fig. 1) that is removed during cross-bridge formation by a transpeptidase (11). In some cases, the cross bridges are not formed and the terminal D-alanine may or may not be removed by a carboxypeptidase (11).Zoocin A is a streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881. The genes for zoocin A and for the zoocin A immunity factor (zif) are adjacent on the chromosome and are divergently transcribed (1). Zoocin A hydrolyzes the cell walls of some closely related streptococci, including S. pyogenes and S. mutans, the main causative agents of group A streptococcal sore throat and dental caries, respectively (20). Zoocin A is presumed to target peptidoglycan cross bridges based on three lines of evidence. First, the catalytic domain of zoocin A has a high degree of similarity to the catalytic domains of several bacteriolytic endopeptidases (lysostaphin [21], ALE-1 [22], LytM [23], and millericin B [3]). Second, the introduction of the gene for lysostaphin resistance into a zoocin A-sensitive streptococcus resulted in the insertion of serines into its peptidoglycan cross bridges and a decrease in sensitivity to zoocin A (6). Third, zoocin A covalently binds penicillin and slowly hydrolyzes a chromogenic cephalosporin, both of which are D-alanyl-D-alanine analogs. These three lines of evidence suggest that zoocin A hydrolyzes the bond between the terminal D-alanine of the stem peptide and the first Lalanine of the cross bridge (8).If zoocin A is an endopeptidase, determination of its site of action on streptococcal peptidoglycans only by the mass of the products is not possible beca...

A panel of rat monoclonal antibodies was used to classify mouse mast cells from four different sources on-the basis of their surface determinants. Three populations of mast cells were derived by culturing normal bone marrow, spleen, and peripheral blood cells, whereas the fourth population was isolated directly from the peritoneal cavity. For each population, a surface antigen phenotype was determined by indirect immunofluorescence microscopy and compared to the phenotypes of the other populations of mast cells. The results showed that culture-derived mast cells, regardless of origin, had nearly identical antigen phenotypes, suggesting that these populations were comparable. By contrast, per-itoneal mast cells had a different phenotype, suggesting that they represented a class distinct from culture-derived mast cells. These findings, which corroborate classification schemes based on nonimmunologic methods, demonstrate that subpopulations of mouse mast cells can be distinguished antigenically.Mast cell heterogeneity has been observed in vivo for many years, based principally on histologic and ultrastructural criteria (1-4). More recently, biochemical and functional characterizations of-mast cells (5, 6) have confirmed that two major classes of mast cells exist in vivo, one found in the skin, con--nective tissue, and peritoneal cavity and the other in the intestinal mucosa.The ability to culture mast cells in vitro from progenitor cells in hematopoietic (7)(8)(9)(10)(11) and lymphoid (12, 13) tissues has provided readily available populations of mast cells for phenotypic characterization and comparison with mast cells found-in vivo. Cultured mast cells resemble mucosal mast cells in several ways, including histamine content (5, 10) and dependence on a factor(s) from thymus (14) or thymus-derived lymphocytes (10) for growth. Culture-derived mast cells differ from connective tissue mast-cells in these same-characteristics. These two populations also possess distinct glycosaminoglycans (15, 16) and elaborate different metabolites of arachidonic acid (17).Heterogeneity also could-be expressed by mast cells through their surface determinants. Accordingly, we used rat monoclonal antibodies made against cultured, adherent mouse bone marrow cells to compare various mast cell.populations on the -basis of their surface antigen phenotypes. We show here that these -reagents are useful for the classification of mouse mast cells.

The effect of tunicamycin, which inhibits N-linked glycosylation, on the replication of Epstein-Barr virus was examined. Tunicamycin markedly reduced the yield of virus from producing cells. At concentrations of 1 to 2 jig of tunicamycin per ml, there was a buildup of intracellular virus in P3HR1-CI13 cells but not in MCUV5 cells; at a concentration of 5 jig of tunicamycin per ml in P3HR1-CI13 cells, viral DNA synthesis was inhibited as well. Viral glycoproteins lacking N-linked sugars were apparently inserted into the cell membrane, and the small amount of virus made in the presence of drug was able to bind specifically to its receptor on B cells. However, the ability of the virus to induce immunoglobulin secretion by fresh human lymphocytes was impaired. This implies a role for viral glycoproteins in the penetration as well as the attachment of virus.

Aerobic cultures of Staphylococcus simulans biovar staphylolyticus characteristically achieved about 17 times higher bacterial densities and produced about 7 times higher concentrations of exoprotein than did anaerobic cultures. However, total exoprotein secreted per unit of bacterial dry weight typically was 2.3 times greater for anaerobic cultures. As determined by SDS-PAGE, anaerobic cultures also produced a wider variety of exoproteins than did aerobic cultures. Three exoenzymes, a staphylolytic endopeptidase, a micrococcolytic hexosaminidase and a thiol protease, were completely repressed during anaerobic growth, which is further evidence for coordination of their production.

Resistance to lysostaphin, a staphylolytic glycylglycine endopeptidase, is due to a FemABX-like immunity protein that inserts serines in place of some glycines in peptidoglycan cross bridges. These modifications inhibit both binding of the recombinant cell wall targeting domain and catalysis by the recombinant catalytic domain of lysostaphin.

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a D-alanyl-L-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three L-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.