Figure 5. In vivo functions of RbpA NTT and CD.
(A) Morphologic effects of RbpA truncations. Msm expressing either RbpAwt, RbpACD-BL-SID, or RbpABL-SID were stained with Hoechst (DNA) and FM 4–64 (membranes), and viewed by both fluorescence illumination and DIC imaging. Representative images are shown. The yellow arrowheads indicate aberrant cell morphology with condensed nucleoid in the RbpABL-SID strain. The white arrowhead indicates an anucleate filament. Quantitation of nucleoid morphology in WT (n = 208) and RbpABL-SID (n = 248) cells is graphed below the image as described in the text. Error bars are 99% confidence interval and p<0.0001 by chi squared test. (B) Unsupervised clustering of RNA-seq gene expression data from triplicate RNA samples from cells expressing RbpAwt, RbpACD-BL-SID, or RbpABL-SID. The cluster was generated from the 200 genes with the greatest variance between strains. The strains are clustered across the top of the heat map and genes clustered to the left of the heat map. (C) Distinct gene expression signatures associated with RbpA domains. Scatterplot of gene expression comparing the log2 fold change of RbpACD-BL-SID (X axis), to RbpABL-SID (Y axis) each compared to wild type cells. All colored points represent genes with statistically significant differences in RNA level compared to WT (adjusted p<0.01) with a fold change of >2 (log2 FC of >1 or <-1). The genes are classified by color according to their expression pattern as follows: orange (overexpressed in both strains), green (underexpressed in both strains), yellow (overexpressed in BL-SID but not CD-BL-SID), pink (underexpressed in BL-SID but not CD-BL-SID), red (overexpressed in CD-BL-SID but not BL-SID), cyan (underexpressed in CD-BL-SID but not BL-SID). Points with dark outline indicate genes in which the difference in fold change between the two strains is two fold or greater. The single dark blue and purple points represent genes in which the difference between strains is >2 fold but in opposite direction. See Supplementary file 7 for gene lists corresponding to each color class.