Applied and Environmental Microbiology, January 2001, p. 251-259, Vol. 67, No. 1
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.251-259.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695-7624
Received 20 July 2000/Accepted 18 October 2000
A novel system that leaks
-galactosidase (
-gal) without a
requirement for secretion or export signals was developed in
Lactococcus lactis by controlled expression of integrated
phage holin and lysin cassettes. The late promoter of the lytic
lactococcal bacteriophage
31 is an 888-bp fragment
(P15A10) encoding the transcriptional activator. When a
high-copy-number P15A10::lacZ.st
fusion was introduced into L. lactis strains C10, ML8,
NCK203, and R1/r1t, high levels of the resultant
-gal activity were
detected in the supernatant (approximately 85% of the total
-gal
activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed
that the phenotype resulted from expression of Tac31A from the
P15A10 fragment, which activated a homologous late promoter
in prophages harbored by the lactococcal strains. Despite the high
levels of
-gal obtained in the supernatant, the growth of the
strains was not significantly affected, nor was there any evidence of
severe membrane damage as determined by using propidium iodide or
transmission electron microscopy. Integration of the holin-lysin
cassette of phage r1t, under the control of the phage
31 late
promoter, into the host genome of MG1363 yielded a similar "leaky"
phenotype, indicating that holin and lysin might play a critical role
in the release of
-gal into the medium. In addition to
-gal,
tetanus toxin fragment C was successfully delivered into the growth
medium by this system. Interestingly, the X-prolyl dipeptidyl
aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not
delivered at significant levels outside the cell. These findings point
toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known
secretion and export signals.
Paper no. FSR00-20 of the Department of Food Science, Southeast
Dairy Foods Research Center, North Carolina State University, Raleigh,
N.C.
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