Juan Santana and Matthew Jorgenson will Present Their Research on October 24, 2013
ABSENCE OF THE DROSOPHILA MYB ONCOPROTEIN, BUT NOT ITS INTERACTING PARTNER NURF, RESULTS IN CELL CYCLE PROGRESSION DEFECTS
c–Myb is a proto-oncogene which when mutated causes leukemias and lymphomas in birds and mammals. Vertebrates contain three representatives of the Myb gene family consisting of A-, B- and c-Myb, all of which encode DNA-binding factors that are important for the proper expression of large numbers of genes including those that regulate cell cycle progression. Drosophila melanogaster contains a single Myb gene (Dm-Myb), mutants of which die before reaching adulthood. Dm–Myb protein is present in a complex which includes the nucleosome remodeling factor NURF. Through yeast two-hybrid experiments andgenetic screens, we have shown that Dm–Myb is directly interacting with the major subunit of NURF (NURF301). In light of these results, we performed gene expression analyses in wing discs of Dm-Myb and Nurf301 mutant animals under the assumption that a significant number of genes are co-regulated by both proteins. As expected but nonetheless striking, there is a strong overlap of the genes regulated by these two proteins. We now show that in vivo, as previously reported in cell lines, Dm-Myb is necessary for the activation of cell cycle genes, specifically those involved in the G2/M transition. Interestingly, despite the strong overlap of genes co-regulated by Dm-Myb and NURF, the latter is not required for the regulation of this class of genes, suggesting that Dm-Myb and NURF function together in some contexts but independently in others. Consistent with these data, Dm-Myb, but not Nurf301, mutants have an increased mitotic index due to cells arrested in G2/M, which translates into a significant developmental delay in only the Dm-Myb mutants.
RlpA is a peptidoglycan hydrolase that facilitates daughter cell separation in Pseudomonas aeruginosa
RlpA is a protein of heretofore unknown function found in many Gram-negative bacteria. In Escherichia coli RlpA localizes to the septal ring, suggesting the protein is involved in cell division, but mutants of rlpA have no obvious phenotype (Gerding et al, J. Bacteriol. 2009; Arends et al, J. Bacteriol. 2010). The sequence of RlpA indicates it is an outer membrane lipoprotein with a double-psi beta barrel (DPBB) domain and a peptidoglycan-binding SPOR domain. We report here that E. coli RlpA is present at about 600 molecules per cell and traffics to the outer membrane, as predicted. Because we could not find an rlpA-related phenotype in E. coli, we turned to Pseudomonas aeruginosa, for which we had access to an ordered library of transposon mutants (Liberati et al, PNAS 2006). Interestingly, an rlpA::Tn mutant formed chains of 4-8 cells when grown in media of low osmotic strength. We confirmed the chaining phenotype by constructing an in-frame deletion (ΔrlpA). Both mutants could be rescued by complementation with rlpA or an rlpA-mCherry fusion. The RlpA-mCherry fusion protein localized to the midcell during cell division and localization required the SPOR domain. Analysis of purified peptidoglycan from the ΔrlpA mutant by HPLC revealed an increase in a muropeptide whose structure was determined to be a tetrasaccharide (GlcNAc-MurNAc-GlcNAc-MurNAc) by amino acid/amino sugar analysis and mass spectrometry. Purified RlpA protein hydrolyzed the tetrasaccharide moiety from intact peptidoglycan purified from the ΔrlpA mutant, but did not hydrolyze the purified tetrasaccharide fragment. RlpA proteins with amino acid substitutions in the DPBB had little or no detectable cell wall hydrolysis activity and were not very effective at rescuing daughter cell separation in vivo. We conclude that RlpA is peptidoglycan hydrolase that cleaves the glycosidic linkage between GlcNAc and MurNAc in the peptidoglycan strands to facilitate daughter cell separation.