Affiliation:
1. Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas, USA
Abstract
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, Tn
925
Orf14, and pIP501 TraG, partially complemented the Δ
prgK
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
38 articles.
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