The Bacillus subtilis GlpP protein, antitermination and mRNA stability

Detta är en avhandling från Department of Microbiology, Lund University

Sammanfattning: Bacillus subtilis can grow on glycerol or glycerol-3-phosphate (G3P) as the sole carbon and energy source. The glpD gene encodes G3P dehydrogenase which is required for the catabolism of glycerol and G3P. In the non-coding leader sequence of glpD there is an inverted repeat followed by a run of AT basepairs which has been shown to function as a Rho-independent terminator (a stem-loop structure followed by a run of U residues). The glpP gene encodes an antiterminator protein which together with G3P induces readthrough of the terminator. GlpP mutants cannot grow on glycerol or G3P. Revertants of GlpP mutants that could grow on glycerol were studied. A majority of them carried suppressor mutations in the inverted repeat of the glpD leader and expressed glpD constitutively. Some of the revertants had a temperature sensitive (ts) phenotype, i.e. they grew on glycerol at 32oC but not at 45oC. This was shown to be due to a destabilisation of their glpD mRNA. The mRNA was stabilised in the presence of GlpP. Expression of glpD is subject to glucose repression but in the above revertants it is glucose insensitive. Introduction of a functional glpP gene into the revertants restored glucose repression, indicating that GlpP is the mediator. When the glpD leader and the glpP gene were transferred to E. coli, expression from the glpD leader was subject to catabolite repression which supports the notion that GlpP is important for this control. Two non ts revertants were studied. Their mutations, a point mutation and a 8 bp duplication, respectively, were situated in the sequence of the glpD leader that corresponds to the stem of the terminator, whereas the mutations of the ts revertants involve the loop. glpD transcripts from the non ts revertants had longer half-lives than wild-type and ts glpD transcripts in the presence of GlpP. GlpP did not increase the half-lives of the non ts glpD transcripts. The glpD leader carrying the point mutation could not titrate GlpP in vivo which indicates that the mutation interferes with binding of GlpP. The GlpP protein was His-tagged and purified. It bound specifically to glpD leader mRNA in native gel shifts. Specific binding of GlpP to glpD leader mRNA in vivo was demonstrated in titration experiments. Fusions between different glpD leaders and lacZ were made. Transcripts from these fusions had the same stability as the corresponding glpD transcript, proving that the glpD leader is a major stability determinant for the fusion transcripts. glpD leader-lacZ fusions were integrated into the E. coli chromosome and steady state levels and half-lives of the fusion transcripts were determined. Transcripts from a ts glpD leader were considerably more stable in E. coli than in B. subtilis. GlpP caused antitermination also in E. coli but it had no stabilising effect of glpD leader mRNA as it has in B. subtilis. The results point to differences in the mRNA degrading machinery of the two bacteria.

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