PMID- 10816506
OWN - NLM
STAT- MEDLINE
DCOM- 20000623
LR  - 20220408
IS  - 0019-9567 (Print)
IS  - 1098-5522 (Electronic)
IS  - 0019-9567 (Linking)
VI  - 68
IP  - 6
DP  - 2000 Jun
TI  - Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis 
      subsp. cremoris using a new shuttle vector.
PG  - 3516-22
AB  - Staphylococcus aureus harbors redundant adhesins mediating tissue colonization 
      and infection. To evaluate their intrinsic role outside of the staphylococcal 
      background, a system was designed to express them in Lactococcus lactis subsp. 
      cremoris 1363. This bacterium is devoid of virulence factors and has a known 
      genetic background. A new Escherichia coli-L. lactis shuttle and expression 
      vector was constructed for this purpose. First, the high-copy-number lactococcal 
      plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that 
      could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were 
      inserted at one end of the pOri253 multicloning site. Gene expression was 
      assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) 
      expressed luciferase constitutively at a level 10,000 times greater than did the 
      P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal 
      clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. 
      Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA 
      protein attached to their cell walls. This was indicated both by the presence of 
      the protein in Western blots of solubilized cell walls and by the ability of 
      ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive 
      lactococci had clumping titers (titer of 4,112) similar to those of S. aureus 
      Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. 
      These experiments provide a new way to study individual staphylococcal pathogenic 
      factors and might complement both classical knockout mutagenesis and modern in 
      vivo expression technology and signature tag mutagenesis.
FAU - Que, Y A
AU  - Que YA
AD  - Division of Infectious Diseases, Department of Internal Medicine, Centre 
      Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland.
FAU - Haefliger, J A
AU  - Haefliger JA
FAU - Francioli, P
AU  - Francioli P
FAU - Moreillon, P
AU  - Moreillon P
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Infect Immun
JT  - Infection and immunity
JID - 0246127
RN  - 0 (Coagulase)
RN  - 0 (Recombinant Proteins)
RN  - 9001-32-5 (Fibrinogen)
SB  - IM
MH  - Bacterial Adhesion
MH  - Coagulase/*biosynthesis/genetics
MH  - Fibrinogen
MH  - Genes, Bacterial
MH  - *Genetic Vectors
MH  - Lactococcus lactis/*genetics
MH  - Recombinant Proteins/*biosynthesis
MH  - Staphylococcus aureus/enzymology/*genetics/pathogenicity
PMC - PMC97637
EDAT- 2000/05/19 09:00
MHDA- 2000/07/06 11:00
PMCR- 2000/06/01
CRDT- 2000/05/19 09:00
PHST- 2000/05/19 09:00 [pubmed]
PHST- 2000/07/06 11:00 [medline]
PHST- 2000/05/19 09:00 [entrez]
PHST- 2000/06/01 00:00 [pmc-release]
AID - 1703 [pii]
AID - 10.1128/IAI.68.6.3516-3522.2000 [doi]
PST - ppublish
SO  - Infect Immun. 2000 Jun;68(6):3516-22. doi: 10.1128/IAI.68.6.3516-3522.2000.