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ABSTRACTFrancisella tularensisis the bacterial pathogen that causes tularemia in humans and a number of animals. To date, there is no approved vaccine for this widespread and life-threatening disease. The goal of this study was to identify F. tularensismutants that can be used in the development of a live attenuated vaccine. We screened F. novicidatransposon mutants to identify mutants that exhibited reduced growth in mouse macrophages, as these cells are the preferred host cells of Francisellaand an essential component of the innate immune system. This approach yielded 16 F. novicidamutants that were 100-fold more attenuated for virulence in a mouse model than the wild-type parental strain. These mutants were then tested to determine their abilities to protect mice against challenge with high doses of wild-type bacteria. Five of the 16 attenuated mutants (with mutations corresponding to dsbB, FTT0742, pdpB, fumA, and carBin the F. tularensisSCHU S4 strain) provided mice with protection against challenge with high doses (>8 × 105CFU) of wild-type F. novicida. We believe that these findings will be of use in the design of a vaccine against tularemia.

ABSTRACTDegradation of Saccharomyces cerevisiaeG1cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCFGrr1). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (ΔN-Sic1), a substrate of SCFCdc4, results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to ΔN-Sic1 switches degradation of Sic1 from SCFCdc4to SCFGrr1. ΔN-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCFCdc4target for SCFGrr1-mediated degradation by the ubiquitin-proteasome pathway.

Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.