Abstract:
Zoocin A is a bacteriolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881 that is able to hydrolyze the cell walls of some streptococcal species. The site of action on peptidoglycan by zoocin A was previously unknown. To determine its site of action, susceptible peptidoglycan from a zooA-zif knockout strain was hydrolyzed with mutanolysin and zoocin A to create muropeptides that could be separated by reverse phase-high pressure liquid chromatography (RP-HPLC) and analyzed by mass spectrometry (MS). The muropeptide structures could not be confirmed by MS alone due to the composition of streptococcal peptidoglycans. Therefore, the generated muropeptides were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by MS in the negative-ion mode, which simplified analysis. Zoocin A was shown to hydrolyze the bond between the terminal D-alanine of the stem peptide and the N-terminal L-alanine of the cross bridge of susceptible peptidoglycan. Producer cell immunity to zoocin A is due to Zif, the zoocin A immunity factor, which is encoded adjacent to zoocin A on the chromosome of Streptococcus equi subsp. zooepidemicus 4881. Zif is a member of the FemABX family of proteins, which are non-ribosomal peptidyl transferases that add the cross bridge amino acids during peptidoglycan synthesis. In streptococci, MurM and MurN are responsible for this addition, and Zif is highly similar to MurN. To determine how Zif modifies peptidoglycan to make it resistant to zoocin A, peptidoglycan from strains with and without zif were purified and hydrolyzed by the streptococcolytic phage lysin B30, which acts both as a muramidase and as a D-alanyl-L-alanine endopeptidase. The generated muropeptides were separated using RP-HPLC and analyzed using SPITC-labeling and tandem MS in the negative-ion mode. It was determined that Zif inserts an L-alanine into the peptidoglycan cross bridge, similar to MurM and MurN. Therefore, strains with zif contained cross bridges with mostly three L-alanines instead of mostly two L-alanines. This modification inhibited both the catalytic activity of zoocin A and its ability to bind to peptidoglycans.
