Mutational and Phylogenetic Analyses of the Mycobacterial mbt Gene Cluster

Abstract

The mycobactin siderophore system is present in many Mycobacterium species, including M. tuberculosis and other clinically relevant mycobacteria. This siderophore system is believed to be utilized by both pathogenic and nonpathogenic mycobacteria for iron acquisition in both in vivo and ex vivo iron-limiting environments, respectively. Several M. tuberculosis genes located in a so-called mbt gene cluster have been predicted to be required for the biosynthesis of the core scaffold of mycobactin based on sequence analysis. A systematic and controlled mutational analysis probing the hypothesized essential nature of each of these genes for mycobactin production has been lacking. The degree of conservation of mbt gene cluster orthologs remains to be investigated as well. In this study, we sought to conclusively establish whether each of nine mbt genes was required for mycobactin production and to examine the conservation of gene clusters orthologous to the M. tuberculosis mbt gene cluster in other bacteria. We report a systematic mutational analysis of the mbt gene cluster ortholog found in Mycobacterium smegmatis. This mutational analysis demonstrates that eight of the nine mbt genes investigated are essential for mycobactin production. Our genome mining and phylogenetic analyses reveal the presence of orthologous mbt gene clusters in several bacterial species. These gene clusters display significant organizational differences originating from an intricate evolutionary path that might have included horizontal gene transfers. Altogether, the findings reported herein advance our understanding of the genetic requirements for the biosynthesis of an important mycobacterial secondary metabolite with relevance to virulence.The obligate human pathogen Mycobacterium tuberculosis, most opportunistic mycobacterial human pathogens (e.g., M. avium), and many nonpathogenic saprophytic mycobacteria (e.g., M. smegmatis) produce a structurally complex salicylic acid-derived siderophore known as mycobactin (MBT) (Fig. 1) (5, 33, 35). MBT has a core scaffold of a proposed nonribosomal peptide-polyketide origin consisting of a hydroxyphenylcapped (methyl)oxazoline moiety linked to an N ε -hydroxylysine residue, which is typically connected to a terminal cyclo-N ε -hydroxylysine by a 4-carbon linker. This core scaffold is decorated with a variable fatty acyl substituent on the N ε of the internal N ε -hydroxylysine residue. Structural variants (carboxymycobactins) with acyl substituents terminating in a carboxylate or a methyl ester are also produced. Interestingly, the core scaffold of MBT is remarkably similar to core scaffolds seen in several compounds-some with interesting pharmacological activities-produced by species of the genus Nocardia (Fig. 1). Nocardia is a saprophytic group of actinomycetes closely related to the mycobacteria and includes species that are increasingly recognized as opportunistic human pathogens (2, 25).Studies with cellular and animal models of mycobacterial infection have established the relevance of the M...

Analyses of MbtB, MbtE, and MbtF Suggest Revisions to the Mycobactin Biosynthesis Pathway in Mycobacterium tuberculosis

“…Subsequent genetic studies confirmed that this gene cluster was associated with MBT production in M. tuberculosis (4). Analyses of the homologous clusters from Mycobacterium smegmatis (3) and Nocardia farcinica (15) confirmed that they were involved in the biosynthesis of MBT and the analog nocabactin NA, respectively, providing additional support for the idea that these gene clusters are involved the production of MBT and MBT-like siderophores.…”

“…Filtration and concentration yielded a colorless clear oil. The crude material was purified on silica with 33:1¡30:1 DCM-methanol containing 0.1% NH 3 Synthesis of N 6 -hydroxy-L-Lys (4). 3 (38 mg, 0.11 mmol) was dissolved in DCM (1 ml) in a vial under N 2 .…”

Self-poisoning of Mycobacterium tuberculosis by interrupting siderophore recycling

“…Our results also show that inactivation of siderophore export and recycling delivers a onetwo punch to Mtb by reducing its capacity to take up iron and by siderophore-mediated self-poisoning. For these reasons, targeting siderophore secretion appears to be advantageous compared with biosynthesis (22,45) because inhibition of siderophore biosynthesis is easily overcome by Mtb using heme, in contrast to siderophore secretion (14). However, when and how Mtb gains access to different iron sources during infection is unclear, as are many components of its iron acquisition mechanisms.…”

Iron Homeostasis in Mycobacterium tuberculosis: Mechanistic Insights into Siderophore-Mediated Iron Uptake

“…The hydroxamic acid groups (N-OH) of the ε-Nhydroxylysines, the phenolate oxygen atom, and the nitrogen atom of the oxazoline moiety (circled in Fig. 1) chelate Fe 3ϩ very effectively, explaining the high affinity of the molecule (ϳ10 30 ) for the oxidized form of the metal ion (8), with poor binding seen with Fe 2ϩ . Figure 1 shows the substitutions at R1 to R5 contributing to the variations among the mycobactins produced by different mycobacterial species.…”