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FIG 1

F. nucleatum ATCC 23726 hemagglutination is inhibited by d -galactose but not by l -arginine. (A) Bacteria incubated with 2% sheep red blood cells (RBC) in the absence and presence of 6 mM d -galactose (Gal) or 50 mM l -arginine (Arg). (B) Erythrocytes incubated without bacteria in the absence and presence of inhibitors. Precipitation of the red blood cells (seen as a red dot) represents a lack of hemagglutination.

Three independent clones were found to be deficient in their ability to hemagglutinate sheep RBCs ( Calvin Klein Woman Lacetrimmed Printed Stretch Lowrise Briefs Offwhite Size L Calvin Klein Outlet Amazing Price Pick A Best For Sale Cheap Price Factory Outlet RwFVv262k0
). Analysis of the transposon location in those mutants determined that all three clones harbored the transposon in the same 11.3-kb gene, corresponding to FN1449, an autotransporter protein in F. nucleatum ATCC 25586 previously termed Fap2 for f usobacterial a poptosis p rotein ( 65 ) in ATCC 23726 ( Looking For Get To Buy Sale Online TOPWEAR Tops Goa Goa Huge Range Of Zkk8C
).

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FIG 2

Isolation of F. nucleatum mutants deficient in hemagglutination. (A) Microtiter plate screening for library clones defective in hemagglutination. (B) Hemagglutination assay with 2-fold dilutions of the hemagglutination-deficient mutants (D22, K25, and K50) compared to wild-type F. nucleatum ATCC 23726 and the randomly selected transposon insertion controls: K151, K174, and J78.

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FIG 3

All three hemagglutination-deficient mutants are defective in Fap2. (A) Locations of transposon insertions in the genes of three hemagglutination-deficient mutants. (B) SDS-PAGE demonstrating the absence of the Fap2 protein bands in membrane proteins extracted from all three hemagglutination-deficient mutants compared to the wild type and the randomly selected mutants J78 and K174. The clear band seen in the MW marker lane indicates 245 kDa.

In order to verify that the mutation affected the Fap2 protein, membrane proteins were extracted from wild-type F. nucleatum , from the three hemagglutination-deficient mutants (D22, K25, and K50), and from randomly selected mutants (J78 and K174) used as controls ( Shopping Online Graphique Militar pants Pink amp; Purple Osklen Clearance Find Great Outlet Free Shipping Low Price Fee Shipping Sale Online Buy Cheap Lowest Price O9r69ybiG
, 65 ) The same high-molecular-mass bands were missing in all three mutants. The corresponding bands from wild-type F. nucleatum were sent for mass spectrometry analysis, which identified peptides belonging to the FN1449 protein of F. nucleatum ATCC 25586 ( Cheap Pictures Freeman TPorter Mens Relaxed Plain or unicolor Jeans Freeman T Porter Outlet 2018 New Sale Sale Online 33Q5o8
).

The Fap2 adhesin involved in hemagglutination is also involved in coaggregation. Similar to hemagglutination, coaggregation of F. nucleatum ATCC 23726 with P. gingivalis is galactose sensitive ( 19 , 66 ). All three hemagglutination-deficient mutants (but not the randomly selected J78 mutant) failed to show galactose-sensitive coaggregation with P. gingivalis ( 100% Guaranteed For Sale Sale Factory Outlet TROUSERS Casual trousers Anne Valérie Hash Clearance New Big Sale Cheap Price Clearance Low Price AQKO7lX
to C ). However, the mutants retained their ability to coaggregate with Streptococcus sanguinis ; this coaggregation is mediated by the fusobacterial arginine-inhibitable RadD adhesin ( Womens Camée Sutra Italien Brief Aubade Free Shipping 2018 Classic For Sale 9wjdlaF
) ( Fig. 4D ).

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