To better understand Mtb, we performed a comparative analysis of 31 organisms from the Tuberculosis Database. We studied the evolution of protein families and metabolic pathways, looked for proteins with evidence Figure 5 New predicted RNAs. a) An example of a new predicted RNA. This is the RNA2 in Table 6 . This figure shows a screenshot from the GenomeView Browser [64] . The light blue bars show the coding regions (Rv1230c and Rv1231); the tan bar shows the conserved region predicted by Gumby [65] ; and the green bar shows the region predicted to fold by Evofold [66] . The yellow and green plots in the center show the RNA-seq data. Green signifies reads from the negative strand, and yellow shows the total reads (positive and negative strands). The multiple alignment is shown on the bottom (darker grey signifies a higher degree of conservation; red signifies no alignment at that position). You can see that this predicted RNA region is conserved through M. avium. The rulers at the top show the gene structure. Small red squares show where stop codons are present all six reading frames, indicating that this intergenic region is unlikely to be a protein-coding region missed in the annotation. b) Northern blots validating four of the new, predicted small RNAs (RNA1, RNA2, RNA3, and RNA9 in Table 6 ). 1 Conserved intergenic regions determined by Gumby. 2 Indicates whether this region is predicted to fold by Evofold. 3 Region in M. smegmatis that aligns with the conserved region in Mtb, and its corresponding RPKM value. 4 Tested experimentally 5 Orientation relative to neighboring genes. The first and last characters give the strands of the flanking genes; the middle character gives the strand for the predicted RNA. of selection, and searched for new noncoding RNAs and transcription factor binding site motifs. The most striking features of our analysis are related to lipid metabolism and its regulation. In addition to observing a general expansion of lipid metabolism genes in the Mycobacteria and Rhodococcus, we observe increased expansions of genes related to saturated fatty acid metabolism in the pathogenic Mycobacteria compared to the soil-dwelling Mycobacteria. We also note differences in evolutionary profiles for catabolic and anabolic lipid metabolism genes, and evidence for positive selection in lipid metabolism genes. The cis-regulatory elements bound by the KstR protein, a known regulator of lipid/cholesterol metabolism, are among the strongest, most highly conserved noncoding signals across the Mycobacteria. Both KstR and its binding sites are highly conserved, appearing at the last common ancestor between Rhodococcus and the Mycobacteria.