A phylogenomic analysis of the Actinomycetales mce operons

Abstract

Background: The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins.

Results: The M. tuberculosis mce loci each include two yrbE and six mce genes, which have homology to ABC transporter permeases and substrate-binding proteins, respectively. Operons with an identical structure were identified in all Mycobacterium species examined, as well as in five other Actinomycetales genera. Some of the Actinomycetales mce operons include an mkl gene, which encodes an ATPase resembling those of ABC uptake transporters. The phylogenetic profile of Mkl orthologs exactly matched that of the Mce and YrbE proteins. Through topology and motif analyses of YrbE homologs, we identified a region within the penultimate cytoplasmic loop that may serve as the site of interaction with the putative cognate Mkl ATPase. Homologs of the exported proteins encoded adjacent to the M. tuberculosis mce operons were detected in a conserved chromosomal location downstream of the majority of Actinomycetales operons. Operons containing linked mkl, yrbE and mce genes, resembling the classic organization of an ABC importer, were found to be common in Gram-negative bacteria and appear to be associated with changes in properties of the cell surface.

Conclusion: Evidence presented suggests that the mce operons of Actinomycetales species and related operons in Gram-negative bacteria encode a subfamily of ABC uptake transporters with a possible role in remodeling the cell envelope.

Figure 1

Schematic representation of the M. tuberculosis H37Rv mce loci. Proximal transcription regulators are colored in purple, yrbE genes in blue, mce genes in green, and genes encoding 'conserved mce-associated proteins' in yellow [44].

Figure 2

Schematic representation of the organization of mce loci in Actinomycetales genomes. Genes encoding proteins belonging to Pfam family PF02470 (Mce) are depicted as green boxes, and to family PF02405 (DUF140) as blue boxes. Dashes indicate gaps in gene numbering.

Figure 3

Conserved proteins encoded in the neighborhood of mce genes in Gram-negative bacteria. Coloring reflects conserved domains identified in the key. Protein families shown are: NBD, an ABC transporter ATPase (IPR003439); DUF140 (IPR003453); Mce (IPR003399); Tol, a Ttg2 toluene tolerance protein (IPR008869); STAS, a domain found in sulfate transporters and anti-sigma factor antagonists (IPR002645); VacJ, a lipoprotein of unknown function (IPR007428); BolA, a possible regulator induced by stress (IPR002634); MurA, UDP-N-acetylglucosamine-1-carboxyvinyltransferase (IPR005750); DUF330 (IPR005586); PqiA, an integral membrane protein inducible by superoxide generators (IPR007498); SAM, an S-adenosyl methionine binding methyltransferase (IPR000051); and ABC2, an ABC-2 type permease (IPR013525).

Figure 4

Phylogenetic tree showing relationship between mce-linked ATPases and mycobacterial orthologs. ATPases encoded within mce operons in Actinomycetales species are colored blue; those in Gram-negative bacterial mce operons are colored green. The sequences most similar to nfa51100, SAV5902 and SCO2422 (indicated in bold), in the Actinomycetales genomes listed in Table 1, were identified by BLASTP searches and included in the tree. All of the best hits from mycobacterial species cluster within the Mkl family and are colored red. For comparison, sequences of all M. tuberculosis H37Rv ATPases of ABC uptake transporters were included [20]. All of the top hits from Actinomycetales that do not possess mce operons are rooted among these non-mce-linked ATPases, as are all of the second hits from mycobacterial species. ORFs are designated by (UniProt gene name | protein name).

Figure 5

Phylogenetic tree of Actinomycetales Mce proteins. A non-redundant set of Mce protein sequences were aligned and an unrooted neighbor-joining tree was computed by MEGA. Coloring corresponds to the classification scheme specified in Table 3. ORFs are designated by [gene locus name | operon number (1–8) and gene position (A-F)]. Where operon orthology cannot be inferred, operons are designated: -1, -2.

Figure 6

Illustration of conserved regions and predicted secondary structure of Actinomycetales Mce proteins. Six separate alignments of the Mce proteins (A-F) listed in Table 3 were submitted to JPred and the consensus secondary structure prediction estimated manually. White boxes represent α-helices and grey arrows β-strands. The C-terminal proline-rich region had low complexity and varied in length from 10–250 amino acids. Signal sequences were identified by SignalP and lipid attachment sites matched the ProSite motif PS00013.

Figure 7

Phylogenetic tree of Actinomycetales YrbE proteins. A non-redundant set of YrbE protein sequences were aligned and an unrooted neighbor-joining tree was computed by MEGA. Coloring corresponds to the classification scheme specified in Table 3. ORFs are designated by [gene locus name | operon number (1–8) and gene position (A, B)]. Where operon orthology cannot be inferred, operons are designated: -1, -2.

Figure 8

Predicted topology and conserved sequence motif of Actinomycetales YrbE proteins. (A) The consensus topology prediction of Actinomycetales YrbE proteins analysis is shown compared to that of a typical ABC permease [42]. (B) WebLogo illustration of the conserved YrbE EExDA sequence motif identified through MEME analysis.

Figure 9

Phylogenetic tree of mycobacterial Mas domain sequences. The conserved Mas domains of mycobacterial proteins listed in Table 4 were aligned and an unrooted neighbor-joining tree was computed by MEGA. Coloring corresponds to the classification scheme specified in Table 3. ORFs are designated by [gene locus name | operon number (1, 3, 4, 7) and gene position (A-D)]. Where operon orthology cannot be inferred, operons are designated: -1, -2.

Figure 10

Representative architectures of Mas domain-containing proteins. Membrane topology predictions for the 66 Mas proteins listed in Table 4 indicated that the conserved domain was located on the extracellular side of the cytoplasmic membrane. The Mas domain was predicted to remain anchored in the majority of proteins (A), but cleaved in eight (B). Three transmembrane segments were identified in seven proteins and four of these were classified as RDD domains (C, D). Five proteins contained an N-terminal coiled-coil region (E), and one, a serine-threonine protein kinase domain (STPK; F).