PMID:17233731

Citation	Paradis-Bleau, C, Cloutier, I, Lemieux, L, Sanschagrin, F, Laroche, J, Auger, M, Garnier, A and Levesque, RC (2007) Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage phiKZ gp144 lytic transglycosylase. FEMS Microbiol. Lett. 266:201-9
Abstract	The gp144 endolysin gene from the Pseudomonas aeruginosa phage phiKZ 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 alpha-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 phiKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.
Links	PubMed Online version:10.1111/j.1574-6968.2006.00523.x
Keywords	Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Bacteriolysis; Carbohydrate Sequence; Circular Dichroism; Computational Biology; Gene Expression/drug effects; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Molecular Sequence Data; Nitrogen/pharmacology; Peptidoglycan/chemistry; Peptidoglycan/metabolism; Peptidoglycan Glycosyltransferase/genetics; Peptidoglycan Glycosyltransferase/metabolism; Pseudomonas Phages/enzymology; Pseudomonas Phages/genetics; Pseudomonas Phages/growth & development; Pseudomonas aeruginosa/genetics; Pseudomonas aeruginosa/metabolism; Pseudomonas aeruginosa/virology; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPDPK:Q8SD18	involved_in	GO:0044659: cytolysis by virus of host cell	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
BPDPK:Q8SD18		GO:0044659: cytolysis by virus of host cell	ECO:0000314:		P		Decrease in bacterial concentration of E. coli and P. aeruginosa compared to the gram positive controls shows cell lysis by gp144 (Figure 1A).	complete CACAO 10916
BPDPK:Q8SD18		GO:0008933: lytic transglycosylase activity	ECO:0000314:		F		Table 1 shows lytic transglycosylase activity due to the presence of (1,6)-anhydro N-acetylmuramic muropeptides.	complete CACAO 10917
BPDPK:Q8SD18	enables	GO:0008933: lytic transglycosylase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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