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.

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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...

The differential rate of extracellular protein formation by Staphylococcus staphylolyticus, the lysostaphin-producing organism, was biphasic with a low rate of exoprotein secretion during exponential growth and an increased rate during the post-exponential phase of growth. After 20 h, when no further exoprotein was secreted, exoprotein accounted for 5% of the total protein in the culture. The secretion of three extracellular enzymes was monitored and found to represent a constant proportion of total exoprotein at exoprotein concentrations greater than 0.1 mg ml-l.