Comparative analysis of Mycobacterium and related Actinomycetes yields insight into the evolution of Mycobacterium tuberculosis pathogenesis

Abstract

Background: The sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum.

Results: Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four.

Conclusions: Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org.

Figure 1

Phylogenetic Tree based on uniform SYNERGY orthogroups, scaled by phylogenetic distance. The labels A-C indicate the branches selected for further analysis in our d_N/d_S analysis (A: The branch leading to the Mtb cluster; B: The branch leading to the pathogenic Mycobacteria; C: The branch leading to the non-pathogenic, soil-dwelling Mycobacteria). See Methods for details on the phylogenetic tree construction.

Figure 2

Summary of SYNERGY results: Number of gains, losses, and duplication at each node. For each node, the node number is marked in black; the total number of genes present at each node is indicated in red, and the numbers of gains, losses, and duplications are indicated in parenthesis in blue http://www.broadinstitute.org/ftp/pub/seq/msc/pub/SYNERGY/index.html.

Figure 3

Evolution of gene categories. This figure shows several examples of the evolution of metabolic pathways, PFAM domains, and GO term descriptors. Graphics similar to these can be found for each category in the supplementary information website at http://www.broadinstitute.org/ftp/pub/seq/msc/pub/SYNERGY/index.html. a) Fatty acid degradation metabolic pathway. b) PFAM group PF00823 (PPE genes). c) PFAM group PF00440 (tetR family transcription factors). d) GO term 0009405 (pathogenesis.)

Figure 4

Evolution of genes upregulated when grown on saturated or unsaturated fatty acids. Genes upregulated by at least 1.5 standard deviations are indicated here. a) Genes expressed under palmitic acid but not oleic or linoleic (genes expressed in saturated fatty acid conditions). b) Genes expressed under linoleic or oleic acid but not palmitic (genes expressed under unsaturated fatty acid conditions). c) The ratio of the phylogenetic profiles for genes expressed under palmitic and linoleic acid, normalized by genome size.

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).

Figure 6

New predicted motif with binding sites upstream of fatty acid-related genes. a) Motif logo. b) Conserved binding site locations for this new motif are marked with red x's. Red lines indicate orthologous relationships between genes in Mtb H37Rv, M. avium 104, and M. smegmatis.