Affiliation:
1. Department für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1180 Vienna, Austria
Abstract
ABSTRACT
The gram-positive bacterium
Paenibacillus alvei
CCM 2051
T
is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β-
d
-Gal
p
-(1[α-
d
-Glc
p
-(1→6)]→4)-β-
d
-Man
p
NAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO
2
→4-β-
d
-Man
p
NAc-(1→4)]→3)-α-
l
-Rha
p
-(1→3)-α-
l
-Rha
p
-(1→3)-α-
l
-Rha
p
-(1→3)-β-
d
-Gal
p
-(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the
sgsE
S-layer gene promoter of
Geobacillus stearothermophilus
NRS 2004/3a and on the
Geobacillus-Bacillus-Escherichia coli
shuttle vector pNW33N. As a target gene,
wsfP
, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional
P. alvei
CCM 2051
T
wsfP
mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of
wsfP
in the glycan-deficient
P. alvei
mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism
P. alvei
CCM 2051
T
.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Reference40 articles.
1. Abu-Qarn, M., J. Eichler, and N. Sharon. 2008. Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea. Curr. Opin. Struct. Biol.18:544-550.
2. Altman, E., J. R. Brisson, P. Messner, and U. B. Sleytr. 1991. Structure of the glycan chain from the surface layer glycoprotein of Bacillus alvei CCM 2051. Biochem. Cell Biol.69:72-78.
3. Altman, E., C. Schäffer, J.-R. Brisson, and P. Messner. 1995. Characterization of the glycan structure of a major glycopeptide from the surface layer glycoprotein of Clostridium thermosaccharolyticum E207-71. Eur. J. Biochem.229:308-315.
4. Ash, C., F. G. Priest, and M. D. Collins. 1993. Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek64:253-260.
5. Construction of an Alpha Toxin Gene Knockout Mutant of
Clostridium perfringens
Type A by Use of a Mobile Group II Intron
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