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. 2007 Jul;189(13):4809-14.
doi: 10.1128/JB.01786-06. Epub 2007 Apr 27.

YbxF, a protein associated with exponential-phase ribosomes in Bacillus subtilis

Ludĕk Sojka  1 , Vladimír Fucík, Libor Krásný, Ivan Barvík, Jirí Jonák
Affiliations

YbxF, a protein associated with exponential-phase ribosomes in Bacillus subtilis

Ludĕk Sojka et al. J Bacteriol. 2007 Jul.

Abstract

The ybxF gene is a member of the streptomycin operon in a wide range of gram-positive bacteria. In Bacillus subtilis, it codes for a small basic protein (82 amino acids, pI 9.51) of unknown function. We demonstrate that, in B. subtilis, YbxF localizes to the ribosome, primarily to the 50S subunit, with dependence on growth phase. Based on three-dimensional structures of YbxF generated by homology modeling, we identified helix 2 as important for the interaction with the ribosome. Subsequent mutational analysis of helix 2 revealed Lys24 as crucial for the interaction. Neither the B. subtilis ybxF gene nor its paralogue, the ymxC gene, is essential, as shown by probing DeltaybxF, DeltaymxC, or DeltaybxF DeltaymxC double deletion strains in several functional assays.

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Figures

FIG. 1.
FIG. 1.
Streptomycin operon of B. subtilis. Genes belonging to the operon are colored dark gray. Black arrows indicate promoters; transcription terminators are shown as stem-loop structures.
FIG. 2.
FIG. 2.
Growth curves of B. subtilis ΔybxF (×) and wild-type (▴) strains (Table 1) in Spizizen rich (A) and Spizizen minimal (B) media with incubation at 37°C.
FIG. 3.
FIG. 3.
Western blot analysis of the B. subtilis sonicated cells of strain II (ΔybxF ybxF-gfp), IV (ΔybxF gfp), and 168 (ybxF+ parent strain [P]). M, marker.
FIG. 4.
FIG. 4.
GFP fluorescence of B. subtilis cells. Exponentially growing cells of strain IV (ΔybxF gfp) expressing GFP only (A) and exponentially growing cells of strain II (ΔybxF ybxF-gfp) expressing the YbxF-GFP fusion protein (B). Arrows indicate the GFP signal localized predominantly toward the cell poles.
FIG. 5.
FIG. 5.
GFP fluorescence of the B. subtilis ribosomes. Ribosomes were isolated from exponentially growing cells; GFP fluorescence of strain II (ΔybxF ybxF-gfp) ribosomes isolated from the stationary phase is also shown.
FIG. 6.
FIG. 6.
Ribosomal subunit profile and Western blot analysis of ribosome fractions of exponentially growing strain II (ΔybxF ybxF-gfp) using GFP antibody. C+, ribosomes of strain II (ΔybxF ybxF-gfp) isolated from exponentially growing cells.
FIG. 7.
FIG. 7.
3D homology model of YbxF obtained using as a template wheat germ L30e (PDB entry 1YSH, chain C). Basic amino acid residues of the second α-helix are indicated.
FIG. 8.
FIG. 8.
GFP fluorescence of B. subtilis ribosomes isolated from exponentially growing cells of the strains with mutated YbxF protein.
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