Open AccessPeptidoglycan lytic activity of the Pseudomonas aeruginosa phage φKZ gp144 lytic transglycosylase
Author(s) -
ParadisBleau Catherine,
Cloutier Isabelle,
Lemieux Lise,
Sanschagrin François,
Laroche Jérôme,
Auger Michèle,
Garnier Alain,
Levesque Roger C.
Publication year - 2007
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2006.00523.x
Subject(s) - lytic cycle , peptidoglycan , lysin , lysis , escherichia coli , biochemistry , biology , bacterial cell structure , calcein , bacteria , pseudomonas aeruginosa , microbiology and biotechnology , membrane , chemistry , bacteriophage , enzyme , gene , virus , genetics , virology
The gp144 endolysin gene from the Pseudomonas aeruginosa phage φKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram‐negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN 3 ), an inhibitor of the protein export machinery, and into an E. coli MM52 secA ts mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in α‐helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from φKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.