The Bacillus subtilis GTP Binding Protein Obg and Regulators of the ς B Stress Response Transcription Factor Cofractionate with Ribosomes

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Abstract
Obg, an essential GTP binding protein of Bacillus subtilis , is necessary for stress activation of the ς B transcription factor. We investigated Obg's cellular associations by differential centrifugation of crude B. subtilis extracts, using an anti-Obg antibody as a probe to monitor Obg during the fractionation, and by fluorescent microscopy of a B. subtilis strain in which Obg was fused to green fluorescent protein. The results indicated that Obg is part of a large cytoplasmic complex. In subsequent analyses, Obg coeluted with ribosomal subunits during gel filtration of B. subtilis lysates on Sephacryl S-400 and specifically bound to ribosomal protein L13 in an affinity blot assay. Probing the gel filtration fractions with antibodies specific for ς B and its coexpressed regulators (Rsb proteins) revealed coincident elution of the upstream components of the ς B stress activation pathway (RsbR, -S, and -T) with Obg and the ribosomal subunits. The data implicate ribosome function as a possible mediator of the activity of Obg and the stress induction of ς B .