PMID:24244005

Citation	Laverde Gomez, JA, Bhatty, M and Christie, PJ (2014) PrgK, a multidomain peptidoglycan hydrolase, is essential for conjugative transfer of the pheromone-responsive plasmid pCF10. J. Bacteriol. 196:527-39
Abstract	Peptidoglycan (PG) hydrolases associated with bacterial type IV secretion systems (T4SSs) are thought to generate localized lesions in the PG layer to facilitate assembly of the translocation channel. The pheromone-responsive plasmid pCF10 of Enterococcus faecalis encodes a putative cell wall hydrolase, PrgK, and here we report that a prgK deletion abolished functionality of the pCF10-encoded T4SS as monitored by pCF10 conjugative transfer. Expression in trans of wild-type prgK fully complemented this mutation. PrgK has three potential hydrolase motifs resembling staphylococcal LytM, soluble lytic transglycosylase (SLT), and cysteine-, histidine-dependent amidohydrolase/peptidase (CHAP) domains. Complementation analyses with mutant alleles established that PrgK bearing two hydrolase domains in any combination supported near-wild-type plasmid transfer, and PrgK bearing a single hydrolase domain supported at least a low level of transfer in filter matings. When exported to the Escherichia coli periplasm, each domain disrupted cell growth, and combinations of domains additionally induced cell rounding and blebbing and conferred enhanced sensitivity to osmotic shock. Each domain bound PG in vitro, but only the SLT domain exhibited detectable hydrolase activity, as shown by zymographic analyses and release of fluorescent PG fragments. Genes encoding three T4SS-associated, putative hydrolases, Lactococcus lactis CsiA, Tn925 Orf14, and pIP501 TraG, partially complemented the ΔprgK mutation. Our findings establish that PrgK is an essential component of the pCF10-encoded Prg/Pcf T4SS and that its hydrolase domains coordinate their activities for full PrgK function. PrgK is indispensable for plasmid transfer in liquid matings, suggestive of a role in formation or stabilization of mating junctions.
Links	PubMed PMC3911162 Online version:10.1128/JB.00950-13
Keywords	Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Enterococcus faecalis/enzymology; Enterococcus faecalis/genetics; Enterococcus faecalis/metabolism; Gene Expression Regulation, Bacterial/physiology; Gene Expression Regulation, Enzymologic; Mutation; N-Acetylmuramoyl-L-alanine Amidase/classification; N-Acetylmuramoyl-L-alanine Amidase/genetics; N-Acetylmuramoyl-L-alanine Amidase/metabolism; Pheromones/metabolism; Plasmids/metabolism; Protein Structure, Tertiary
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ENTFL:D1LHF5		GO:0009253: peptidoglycan catabolic process	ECO:0000314:		P		Fig. 2,3, and 4 show that PrgK PG hydrolase domains are active in vivo and in vitro.	complete CACAO 10758
ENTFL:D1LHF5	Contributes to	GO:0043684: type IV secretion system complex	ECO:0000315:		C		Fig. 1 shows that PrgK and its PG hydrolase domains are required for conjugation.	complete CACAO 10759

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