Phylogenomic Reappraisal of Fatty Acid Biosynthesis, Mycolic Acid Biosynthesis and Clinical Relevance Among Members of the Genus Corynebacterium

Abstract

The genus Corynebacterium encompasses many species of biotechnological, medical or veterinary significance. An important characteristic of this genus is the presence of mycolic acids in their cell envelopes, which form the basis of a protective outer membrane (mycomembrane). Mycolic acids in the cell envelope of Mycobacterium tuberculosis have been associated with virulence. In this study, we have analysed the genomes of 140 corynebacterial strains, including representatives of 126 different species. More than 50% of these strains were isolated from clinical material from humans or animals, highlighting the true scale of pathogenic potential within the genus. Phylogenomically, these species are very diverse and have been organised into 19 groups and 30 singleton strains. We find that a substantial number of corynebacteria lack FAS-I, i.e., have no capability for de novo fatty acid biosynthesis and must obtain fatty acids from their habitat; this appears to explain the well-known lipophilic phenotype of some species. In most species, key genes associated with the condensation and maturation of mycolic acids are present, consistent with the reports of mycolic acids in their species descriptions. Conversely, species reported to lack mycolic acids lacked these key genes. Interestingly, Corynebacterium ciconiae, which is reported to lack mycolic acids, appears to possess all genes required for mycolic acid biosynthesis. We suggest that although a mycolic acid-based mycomembrane is widely considered to be the target for interventions by the immune system and chemotherapeutics, the structure is not essential in corynebacteria and is not a prerequisite for pathogenicity or colonisation of animal hosts.

Keywords: Corynebacterium; fatty acid chains; mycolic acid biosynthesis; phylogenomic diversity; virulence.

Figure 1

Structural features and sources of mycolic acid components. Mycolic acids are α-alkyl, β-hydroxy branched fatty acids that form the basis of the outer wall permeability barrier or ‘mycomembrane’ that is the defining characteristic feature of the cell wall model originally proposed by Minnikin (1982) for the mycobacteria and other genera that belong to the order Mycobacteriales. Briefly, mycolic acids are generated from the condensation of two fatty acyl chains, known as α-and meromycolyl chains. The α-chain (blue) precursor is activated by carboxylation (position indicated by * and see Figure 5). It then participates in a decarboxylative condensation reaction with the carboxylate group of the meromycolyl chain, the residue of which forms a β-keto group that is subsequently reduced to form the characteristic β-hydroxy group of the mature mycolic acid. There are two potential sources of the fatty acid components of mycolic acids. The bacterium may synthesise them de novo using fatty acid synthase I or acquire them from its habitat. Corynebacteria produce relatively short corynomycolic acids; fatty acids of approx. 16 C chain lengths are used to form both branches. However, in most other genera in Mycobacteriales, the meromycolyl chain is extended by fatty acid synthase II (red chain; the length of R is highly variable but distinctive for each genus and may also contain various structural modifications including double bonds, cyclopropane rings, hydroxy, methoxy, epoxy, wax ester and ketone groups).

Figure 2

A phylogenetic tree from concatenated protein sequence alignment. The scale bar represents amino acid substitution per site. Singleton strains are shown in red.

Figure 3

The distribution of (A) strains from different sources, (B) genome sizes and (C) GC content among different phylogenetic groups. Average size and GC content within the genus are highlighted.

Figure 4

Mycolic acid biosynthetic pathway in Mycobacterium and Corynebacterium. Briefly, mycolic acid biosynthesis proceeds via the activation and condensation of two fatty acyl chains (key enzymes indicated in lower blue box) that ultimately contribute the meromycolyl and α-alkyl chains (see Figure 1). Corynebacteria are unusual in that they do not extend the meromycolyl chain via fatty acid synthase II (FAS-II, key enzymes indicated in middle blue box), but rather condense two short fatty acyl chains that may be generated metabolically via Fatty Acid Synthase I (key enzymes indicated in upper blue box) or taken up from their habitat (route highlighted in green).

Figure 5

Modification of acyl carboxylase activity by β subunit recruitment. Three acyl carboxylase activities are relevant to the production of fatty acids, polyketides and mycolic acids in studied species of Mycobacteriales. All of these are founded on a common α-subunit (AccA3) in a complex with various β-subunits. Those containing the β-subunits AccD4 or AccD5 also contain an ε subunit (AccE5). These acyl carboxylases essentially activate suitable acyl primers to generate substrates for these key biosynthetic processes. A common theme is their involvement in decarboxylative condensation reactions; the carboxyl group is added to promote these reactions and acts as a suitable leaving group (see blue box) that exposes a reactive carbanion that drives the synthetic reaction. The sequence homology shared by these specificity-defining β-subunits is extensive. Their individual functions have been defined through a combination of complex reconstitution and mutagenesis studies. AccD6 prefers an acetyl CoA primer in vitro and the carboxylation reaction provides malonyl CoA, an extension substrate used in the synthesis of fatty acids (including de novo fatty acid synthesis by FAS-I and meromycolyl chain extension by FAS-II) and polyketides. Acyl carboxylase reconstituted with AccD5 and AccE5 prefers a propionyl CoA primer and forms methyl-malonyl CoA which is used in the synthesis of polyketide molecules. The extent of these substrate preferences may vary and appears to influence the conditional essentiality of AccD6; i.e., acyl carboxylase containing AccD5 may be able to generate enough malonyl CoA to support fatty acid biosynthesis in the absence of accD6. AccD4 (likely supported by AccD5 in a heterologous β-subunit complex and AccE5) is responsible for the activation of the α-chain precursor to enable the mycolic condensation (also see Figure 4).