Complete nucleotide sequences of plasmids pACK2 and pACK5 from Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus contains five plasmids, designated pACK1-pACK5. Recently, the nucleotide sequences of three of these plasmids, pACK1, pACK3, and pACK4, were reported. In order to complete the characterization of these five plasmids, the nucleotide sequences of the two remaining plasmids, pACK2 (37683 bp) and pACK5 (3191 bp), were determined. pACK5 is comprised of two regions, one with 85% identity at the nucleotide level to a region of pWBG1773 and another region with an ORF that shares no significant similarity to sequences previously described in GenBank. pACK2 encodes proteins for cadmium resistance and enhanced biofilm formation. The similarities at the nucleotide level among regions of the plasmids of S. simulans bv. staphylolyticus suggest that these plasmids have undergone multiple intermolecular rearrangements.

Inhibition of the activity of both domains of lysostaphin through peptidoglycan modification by the lysostaphin immunity protein.

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.

Complete nucleotide sequences of plasmids pACK1 and pACK3 from Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus contains five plasmids designated pACK1-pACK5. The complete nucleotide sequences of pACK1 (55171bp) and pACK3 (28613bp) were determined and sequence comparison revealed that the entire pACK3 sequence is present on pACK1 (99.98% identical on the nucleotide level) with the sequence unique to pACK1 located between sin and blaZ, which are adjacent in pACK3. The common region contains the staphylococcal beta-lactamase transposon Tn552 and ORFs with similarity to genes encoding a serine-recombinase, enzymes involved in pantothenate biosynthesis, and components of the secA2 region involved in bacterial adherence to host tissues. The common region also contains a cluster of six ORFs that share no significant similarity to sequences previously described in GenBank. The region unique to pACK1, in addition to the genes for lysostaphin and lysostaphin resistance, contains ORFswith similarity to genes encoding a toxin-antitoxin addiction system and proteins involved in plasmid partitioning. The unique region also contains several ORFs that are similar to genes typically found on the chromosome such as those encoding catalase, ferrochelatase, and an enzyme involved in pantothenate biosynthesis. In pACK1, the ends of the unique region contain IS431 elements with direct repeats marking the points where the two plasmid sequences diverge. Several observations suggest that pACK1 was derived by insertion of the unique region into pACK3.

Characterization of pACK4, a mobilizable plasmid from Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus, the lysostaphin-producing organism, contains five plasmids designated pACK1-pACK5. pACK4 was found to be relaxable and to share sequence similarity with a number of well-characterized mobilizable plasmids from other staphylococci. All mobilizable staphylococcal plasmids characterized to date mediate resistance to various antibiotics, but pACK4 is unique because it contains no recognizable antibiotic resistance genes. pACK4 was found to contain an origin of transfer (oriT) region that shares inverted repeat regions and the same nic site as several other mobilizable staphylococcal plasmids. The presence of this conserved oriT region suggested that pACK4 might be mobilized in the presence of a conjugative plasmid. Filter mating studies revealed that pACK4 was mobilized by the conjugative plasmid pGO1. In addition, pACK4 was found to be virtually identical to the recently described plasmid pVGA from Staphylococcus aureus, except that pVGA contains an additional region (vgaA) that confers resistance to pleuromutilin, streptogramin A, and lincosamide. The high sequence similarity among pACK4, pVGA, and several previously described mobilizable staphylococcal plasmids suggests a common origin for these plasmids.

Zif, the zoocin A immunity factor, is a FemABX-like immunity protein with a novel mode of action.

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.

Prevalence and acquisition of the genes for zoocin A and zoocin A resistance in Streptococcus equi subsp. zooepidemicus.

Zoocin A is a streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus strain 4881. The zoocin A gene (zooA) and the gene specifying resistance to zoocin A (zif) are adjacent on the chromosome and are divergently transcribed. Twenty-four S. equi subsp. zooepidemicus strains were analyzed to determine the genetic difference among three previously characterized as zoocin A producers (strains 4881, 9g, and 9h) and the 21 nonproducers. LT-PCR and Southern hybridization studies revealed that none of the nonproducer strains possessed zooA or zif. RAPD and PFGE showed that the 24 strains were a genetically diverse population with eight RAPD profiles. S. equi subsp. zooepidemicus strains 9g and 9h appeared to be genetically identical to each other but quite different from strain 4881. Sequences derived from 4881 and 9g showed that zooA and zif were integrated into the chromosome adjacent to the gene flaR. A comparison of these sequences with the genome sequences of S. equi subsp. zooepidemicus strains H70 and MGCS10565 and S. equi subsp. equi strain 4047 suggests that flaR flanks a region of genome plasticity in this species.

Use of 4-sulfophenyl isothiocyanate labeling and mass spectrometry to determine the site of action of the streptococcolytic peptidoglycan hydrolase zoocin A.

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.

The metal binding site of zoocin A.

Direct metal analysis of the bacteriolytic exoenzyme zoocin A failed to unequivocally identify a putative metal cofactor; hence, indirect experiments utilizing NMR were undertaken to settle this question. Cd(2+) as a surrogate metal ion was reconstituted into EDTA-treated, metal-free recombinant zoocin, and (113)Cd-NMR was employed to explore binding in the protein for this ion. The Cd-substituted enzyme was found to have 80-85% of native streptococcolytic activity. A major (113)Cd resonance at 113.6 ppm was observed which with time split into resonances at 113.6 and 107.2 ppm. A minor (113)Cd resonance at 87.3 ppm was observed which increased in intensity with time. These Cd chemical shifts are indicative of two N atoms and two O atoms ligating directly to the metal site. On the basis of conserved amino acid residues in a homologous protein of known structure, LytM, the ligands in zoocin are tentatively assigned to H45, D49, H133, and some combination of water or buffer ions as the fourth oxygen donor in zoocin A. Comparison of the combined intensities for (113)Cd-substituted zoocin with a known quantity of another Cd-substituted protein gave Cd binding as approximately stoichiometric (1.2+/-0.2) with protein. Additional metal-removal and reconstitution experiments on the recombinant catalytic domain of zoocin implicate Zn(2+) as the metal cofactor. Therefore, the evidence supports zoocin as a single Zn(2+) ion binding metalloenzyme.

Targeting of streptococci by zoocin A.

Zoocin A is a domain-structured peptidoglycan hydrolase produced by Streptococcus equi ssp. zooepidemicus 4881. [(14)C]-zoocin A was used to measure the amount of zoocin A bound to the surface of cells and to purified peptidoglycan. The sensitivity of various streptococci to zoocin A correlated with the amount of zoocin A bound (R(2)=0.8609). Peptidoglycan purified from Streptococcus oralis and Streptococcus rattus were able to bind zoocin A but remained resistant to hydrolysis. All Streptococcus pyogenes strains were extremely sensitive to zoocin A with minimum inhibition concentrations of 31.5 ng mL(-1) or less, suggesting that zoocin A may have potential for use as an enzybiotic.

Plasmid-specified FemABX-like immunity factor in Staphylococcus sciuri DD 4747.

A plasmid from Staphylococcus sciuri DD 4747 had three open reading frames: a replication gene, an N-acetylmuramyl-l-alanine amidase-like gene, and a gene similar to the lysostaphin endopeptidase resistance gene (epr/lif). The epr-like gene was introduced into S. aureus RN4220; the recombinant strain was more resistant to lysostaphin endopeptidase and its cell wall peptidoglycan contained more serines and fewer glycines than the parental strain with the shuttle vector alone. Based on both its function and its similarity to femAB, this gene is a member of the femABX-like immunity gene family. Furthermore, this is the first example of a femABX-like immunity gene that is not linked to the gene for the bacteriolytic enzyme against which it specifies immunity.

The streptococcolytic enzyme zoocin A is a penicillin-binding protein.

Zoocin A is a streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881 that has an unknown site of action on the peptidoglycans of susceptible organisms. Analysis of a mutant strain in which the genes for zoocin A and resistance to zoocin A were inactivated revealed that this strain was more susceptible to beta-lactam antibiotics than the parental organism. Purified zoocin A had weak beta-lactamase activity, bound radioactive penicillin covalently, and its streptococcolytic activity was inhibited by penicillin. Thus, zoocin A is a penicillin-binding protein and presumably is a D-alanyl endopeptidase.

Expression of the genes for lysostaphin and lysostaphin resistance in streptococci.

To determine if the genes for lysostaphin endopeptidase (end) and lysostaphin resistance (epr) function in streptococci, we transferred these genes from Staphylococcus simulans biovar staphylolyticus into two strains of Streptococcus equi subsp. zooepidemicus. The end-containing streptococci were able to produce and process proendopeptidase. Strains containing epr were more resistant to lysis by the streptococcolytic enzyme zoocin A and amino acid analysis of the peptidoglycans of the epr-containing streptococci revealed insertion of serines in their cross bridges. This is the first report of the transfer of a femABX-like immunity factor resulting in a physiologically useful effect in a different genus.