Wall-associated processing of extracellular enzymes of Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus produces a staphylolytic glycylglycine endopeptidase (lysostaphin) and a micrococcolytic endo-beta-N-acetylglucosaminidase (hexosaminidase) as proenzymes that are proteolytically processed through multiple intermediates to their mature forms by an extracellular sulfhydryl protease. Analysis of protease production by immunoblots using antiserum prepared against purified protease and by renaturing activity gels using gelatin as the substrate has revealed that the lysostaphin-processing protease also is produced as a proenzyme, which appears to be autocatalytically processed. Very little proprotease could be detected in supernatants from cultures of S. simulans biovar staphylolyticus, which suggested that the protein was being processed before it was released to the culture medium. Analysis of wall-associated proteins revealed that processing of proprotease occurred primarily in the cell wall. Furthermore, processing of prolysostaphin and prohexosaminidase also occurred in the cell wall matrix.

Zoocin A immunity factor: a femA-like gene found in a group C streptococcus.

A 6.8-kb fragment of Streptococcus equi subsp. zooepidemicus 4881 DNA containing the zoocin A gene (zooA) was cloned in Escherichia coli and sequenced. We have identified a gene we call zoocin A immunity factor (zif), which protects the producer cell from the otherwise lethal action of its own product. Transformation of Streptococcus gordonii DL1 with zooA and zif changed its phenotypic character from a non-zoocin A producing-zoocin A sensitive cell to a zoocin A producing-zoocin A resistant cell. zif has sequence homology to femA (factor essential for methicillin resistance) and lif (lysostaphin immunity factor). No differences were observed in amino acid or amino sugar compositions of peptidoglycan purified from zoocin A sensitive vs. zoocin A immune cells.

Ribotype delineation and description of Staphylococcus sciuri subspecies and their potential as reservoirs of methicillin resistance and staphylolytic enzyme genes.

Three subspecies of Staphylococcus sciuri, S. sciuri subsp. sciuri Kloos, Schleifer, and Smith 1976, 23AL emend. Kloos et al. 1997 [corrected], S. sciuri subsp. carnaticus 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 (70 degrees C) 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. carnaticus 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. carnaticus 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. carnaticus is DD 791 (= ATCC 700058), and the type strain of S. sciuri subsp. rodentium is DD 4761 (= ATCC 700061).

The Lysostaphin Endopeptidase Resistance Gene (epr) Specifies Modification of Peptidoglycan Cross Bridges in Staphylococcus simulans and Staphylococcus aureus.

Volume 61, no. 4, p. 1478, Table 2, column 4: The diameters (in milliliters) of the zones of inhibition for 5-(mu)g methicillin disks given (from top to bottom), "116," "72," "107," and "32," should read "33.5," "22.6," "34.2," and "21.0," respectively. [This corrects the article on p. 1475 in vol. 61.].

The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus.

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 pL150 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.

Plasmid-encoded catalase of Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus contains five plasmids designated pACK1 through pACK5. Non-denaturing electrophoretic analysis of an extract prepared from wild-type cells revealed three bands of catalase activity, whereas an extract of cells cured of pACK1 produced only two catalase bands. Cloning and Southern hybridization analysis showed that there is a catalase structural gene on pACK1. The plasmid-specified catalase was the major activity produced under both aerobic and anaerobic conditions of growth.

Extracellular proteolytic activation of bacteriolytic peptidoglycan hydrolases of Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus secreted two bacteriolytic peptidoglycan hydrolases as proproteins that were activated as they were processed by an extracellular sulphydryl protease. This processing resulted in the production of multiple molecular-mass forms of each enzyme. Cells from early exponential phase cultures were susceptible to lysis by the mature forms of each of the peptidoglycan hydrolases whereas stationary phase cells were resistant. Thus secretion of these bacteriolytic enzymes during early exponential growth as precursors that are activated later by the protease would provide time for the cells to become resistant.

Characteristics of extracellular protein production by a plasmidless derivative of Staphylococcus simulans biovar staphylolyticus.

A derivative of Staphylococcus simulans biovar staphylolyticus cured of all five plasmids present in the wild-type organism was developed, and the characteristics of extracellular protein production by this plasmidless strain were compared to those of the wild type. Although staphylolytic endopeptidase (lysostaphin) and beta-lactamase are known to be plasmid encoded, analysis of this cured strain revealed that most other extracellular proteins are chromosomally encoded.

Beta-lactamase is encoded on plasmid pACK3 in Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus, the lysostaphin-producing organism, contains five plasmids designated pACK1 through pACK5. Hybridization analysis using cloned beta-lactamase gene (bla) as probe and characterization of cured strains revealed that bla resides on pACK3 rather than on the lysostaphin endopeptidase plasmid (pACK1) as reported by others.

Plasmid-encoded lysostaphin endopeptidase resistance of Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus, the lysostaphin-producing organism, secretes a staphylolytic endopeptidase (EC 3.4.99.17) that is encoded on plasmid pACK1. Susceptibility of pACK1-cured strains to lysis by endopeptidase established that resistance to this enzyme is not an inherent property of the organism but rather is encoded on this dispensable plasmid. Furthermore, the enzyme is not an autolysin that is essential for cell wall synthesis because strains lacking the endopeptidase gene grew normally.

Characteristics of extracellular protein production by Staphylococcus simulans biovar staphylolyticus during aerobic and anaerobic growth.

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.

Lack of pleiotropic compensation in extracellular protein production by hypoproducing variants of Staphylococcus simulans biovar staphylolyticus.

The changes in bacterial density, total extracellular protein and activities of three extracellular enzymes were monitored during growth of wild-type Staphylococcus simulans biovar staphylolyticus, a representative pleiotropic variant that produced decreased levels of the three extracellular enzymes, and a variant that produced only 5 of the 14 extracellular proteins secreted by the wild-type organism. Both variants produced less total extracellular protein than did the parental organism. SDS-PAGE of the proteins secreted by these hypoproducing variants showed that all of the extracellular proteins were produced in decreased amounts. No pleiotropic compensation in extracellular protein production was observed for these hypoproducing variants of S. simulans biovar staphylolyticus.

Removal of lipopolysaccharide from acellular Bordetella pertussis vaccine by detergent treatment.

Protective antigen was extracted from Bordetella pertussis cells with 1.0 M NaCl and precipitated with ammonium sulfate, 20-40% saturation (designated fraction 15A-1B). The protective antigen was purified further by detergent (Emulphogene BC720) treatment and adsorption to aluminum hydroxide gel (designated fraction 15A-108A). Compared with B. pertussis vaccine and fraction 15A-1B, fraction 15A-108A retained protective activity as assessed by the mouse protection test, but had reduced protein and markedly reduced endotoxin content. Fraction 15A-108A also had reduced leukocytosis-promoting, histamine sensitizing splenomegaly-inducing, and adjuvant activities. Emulphogene treatment provided a relatively simple method for removing endotoxin from a potential acellular B. pertussis vaccine.

A simple and generally applicable procedure for releasing DNA from bacterial cells.

Treatment of Staphylococcus simulans biovar staphylolyticus cells with acetone before digestion with lysozyme made the cells susceptible to lysis by sodium dodecyl sulfate. This technique was found to be useful for releasing DNA from a wide variety of gram-positive and gram-negative organisms.

Simple screening method for identification of nonpleiotropic mutants for exoenzyme production.

A differential medium that distinguishes between pleiotropic and nonpleiotropic mutants for exoenzyme production has been developed for Staphylococcus simulans biovar staphylolyticus. The medium will facilitate genetic analysis of exoenzyme production by this organism. Generally useful strategies for increasing the sensitivity of indicator plates for detection of exoenzyme activities are presented.

Immunomodulation by Bordetella pertussis: antiviral effects.

Treatment of mice by intraperitoneal inoculation of pertussis vaccine or lipopolysaccharide extracted from B. pertussis will effect resistance to rabies virus, encephalomyocarditis virus, Semliki Forest virus, and Herpes simplex virus. Our previous observations indicated that treatment of C3H/HeN (+/nu) and BDF1 mice with pertussis vaccine injected i.p. five days prior to a mouse adenovirus lethal dose i.p. challenge elicited resistance to clinical disease and death. Susceptibility returned to a portion of the test population 35 days after pertussis vaccine treatment. The pertussis vaccine induced resistance developed in athymic (nude) mice also; however, the population succumbed to infection 35 days later. Titration of pertussis vaccine with respect to induction of resistance indicated the median effective dose (ED50) was approximately 25 micrograms dry weight. This report describes the antiviral activity of acellular components extracted from pertussis vaccine. Extraction of B. pertussis cells with 1.0M NaCl and ammonium sulfate fractionation (20-40% saturation) of the extract resulted in an acellular preparation that induced resistance to lethal dose mouse adenovirus infection. The resistance inducing activity was retained after treatment of the extract with detergent (GAF Emulphogene BC 720) to remove lipopolysaccharide and adsorption to alum gel. Comparison of endotoxin content of pertussis vaccine acellular fractions, polysaccharide fraction and purified lipopolysaccharide suggested that endotoxin probably plays a role in the induction of resistance. The endotoxin content of a Emulphogene-treated preparation that protected 80% of a test population was 39 ng. The lipopolysaccharide extracted from Escherichia coli, Vibrio cholerae, Salmonella typhimurium, and Salmonella minnesota did not induce a resistant state seven days after administration; however, lipopolysaccharide extracted from B. pertussis induced a resistant state.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinate production of three exoenzymes of Staphylococcus staphylolyticus.

Staphylococcus staphylolyticus produced three exoenzymes (a staphylolytic endopeptidase, a hexosaminidase and a protease) coordinately under a range of conditions of induction and repression by various peptides and carbohydrates. Mutants of S. staphylolyticus were isolated and shown to have pleiotropic variations in the production of the three enzymes. Hypo- or hyperproducing mutants of one enzyme were invariably hypo- or hyperproducers for the other two enzymes. Mutants that had lost the ability to produce one of the exoenzymes invariably failed to produce the other two enzymes. Revertants isolated from non-producers that regained the ability to produce one of the exoenzymes always regained the ability to produce the other two as well. These results suggest that the three exoenzymes share a common regulatory or processing mechanism.

The characteristics of extracellular protein secretion by Staphylococcus staphylolyticus.

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-1.