Genome mining for methanobactins

Abstract

Background: Methanobactins (Mbns) are a family of copper-binding natural products involved in copper uptake by methanotrophic bacteria. The few Mbns that have been structurally characterized feature copper coordination by two nitrogen-containing heterocycles next to thioamide groups embedded in a peptidic backbone of varying composition. Mbns are proposed to derive from post-translational modification of ribosomally synthesized peptides, but only a few genes encoding potential precursor peptides have been identified. Moreover, the relevance of neighboring genes in these genomes has been unclear.

Results: The potential for Mbn production in a wider range of bacterial species was assessed by mining microbial genomes. Operons encoding Mbn-like precursor peptides, MbnAs, were identified in 16 new species, including both methanotrophs and, surprisingly, non-methanotrophs. Along with MbnA, the core of the operon is formed by two putative biosynthetic genes denoted MbnB and MbnC. The species can be divided into five groups on the basis of their MbnA and MbnB sequences and their operon compositions. Additional biosynthetic proteins, including aminotransferases, sulfotransferases and flavin adenine dinucleotide (FAD)-dependent oxidoreductases were also identified in some families. Beyond biosynthetic machinery, a conserved set of transporters was identified, including MATE multidrug exporters and TonB-dependent transporters. Additional proteins of interest include a di-heme cytochrome c peroxidase and a partner protein, the roles of which remain a mystery.

Conclusions: This study indicates that Mbn-like compounds may be more widespread than previously thought, but are not present in all methanotrophs. This distribution of species suggests a broader role in metal homeostasis. These data provide a link between precursor peptide sequence and Mbn structure, facilitating predictions of new Mbn structures and supporting a post-translational modification biosynthetic pathway. In addition, testable models for Mbn transport and for methanotrophic copper regulation have emerged. Given the unusual modifications observed in Mbns characterized thus far, understanding the roles of the putative biosynthetic proteins is likely to reveal novel pathways and chemistry.

Figure 1

Post-translational modifications required to produce methanobactins from Methylosinus trichosporium OB3b and Methylocystis rosea SV97T. (A) Mbn from Methylosinus trichosporium OB3b is generated from the precursor peptide MTVKIAQKKVLPVIGRAAALCGSCYPCSCM. Post-translational modifications needed to produce the final natural product include leader peptide cleavage and subsequent N-terminal transamination (orange), oxazolone formation (green), thioamide formation (blue), and disulfide bond formation (yellow). (B) Mbn from Methylocystis rosea SV97T is generated from the precursor peptide MTIRIAKRITLNVIGRASARCASTCAATNG. Post-translational modifications needed to produce the final natural product include leader peptide cleavage, pyrazinedione formation (purple) oxazolone formation (green), thioamide formation (blue), and threonine sulfonation (pink). Several residues that are present in the precursor peptide are missing in the reported structure (gray); the loss of a C-terminal threonine and asparagine had been previously reported, but identification of the precursor peptide indicates that a final glycine is also lost.

Figure 2

The genetic organization of Mbn-producing operons. Genomic regions containing all identified Mbn operons are depicted. Operons are grouped into five families on the basis of operon content, MbnB conservation, and MbnA sequence. All operons contain MbnA (black) and MbnB (orange) and at least one transport protein, a MATE efflux pump (purple), a TonB-dependent transporter (blue) or both. Most operons contain additional biosynthesis-related genes, including MbnC (green), aminotransferases (MbnN, brown) or sulfotransferases (MbnS, dark purple). Additional genes may be related to regulation (MbnR, yellow and MbnI, red) or may play an unknown role in copper homeostasis (MbnP, gold and MbnH, dark blue) Genetic mobility elements of several varieties (light teal) are common in the vicinity of these operons. Metagenomic samples corresponding to unidentified or provisionally identified species are marked (†).

Figure 3

Alignment and classification of the precursor peptide MbnA. (A) A MAFFT alignment of the 20 sequenced MbnA precursors organized by family. Likely leader and core peptides are indicated on the basis of the structurally characterized Mbns and their corresponding MbnA sequences. The alignment is visualized in Jalview and residues are colored following the Taylor color scheme [109], with intensity modified by a conservation color increment of 30%. (B) Logo showing that both leader peptide and core peptide exhibit well-conserved motifs, but the leader peptide is less variant. (C) Phylogenetic tree (constructed in MEGA 5.0 as described in the Methods section) based on a ClustalOmega alignment of the 20 full MbnA. Not pictured in this tree are the three species with structurally characterized Mbns for which no genomic information exists; however, their core peptide sequences indicate that they would be grouped with Methylocystis sp. SC2 and Methylocystis rosea SV97T. Gamma distribution parameter 4.0462.

Figure 4

MbnB: the core Mbn biosynthesis gene. (A) MUSCLE alignment of MbnB, including both domains. Residues are colored following the Taylor color scheme [109], with intensity modified by a conservation color increment of 30%. Truncated metagenomic sequences are not shown in this alignment. (B) Phylogenetic tree (constructed in MEGA 5.0 as described in the Methods section) based on a MUSCLE alignment of all MbnB sequences, not including truncated metagenomic sequences. Gamma distribution parameter 1.5823.

Figure 5

MbnC: the secondary Mbn biosynthesis gene. (A) MUSCLE alignment of MbnC putative biosynthesis proteins. The Group V sequences are divergent and not included. Residues are colored following the Taylor color scheme [110], with intensity modified by a conservation color increment of 30%. Truncated metagenomic sequences are not shown in this alignment. (B) Phylogenetic tree (constructed in MEGA 5.0 as described in the Methods section) based on a MUSCLE alignment of all MbnC sequences, not including truncated metagenomic sequences. Gamma distribution parameter 2.1637.

Figure 6

Proposed Mbn signal transduction pathway featuring the MbnIRT triad. Mbn is secreted from the cell via the MATE multi-drug exporter MbnM and an unknown outer membrane partner. CuMbn is readmitted to the cell via the TonB-dependent transducer MbnT. CuMbn binding to MbnT induces a conformational change that results in contact with both the inner-membrane TonB-ExbD-ExbB complex and MbnR via the unique N-terminal extension of MbnT. CuMbn may or may not enter the cytoplasm intact, but either way, MbnR activates MbnI analogous to a standard FecIRA system. MbnI replaces σ⁷⁰ in the active RNA polymerase complex, activating transcription of Mbn biosynthesis and transport genes, and potentially other operons needed in low copper conditions. In siderophore systems, the negative regulator Fur binds iron as intracellular iron levels rise, and the holo Fur binds to siderophore biosythesis and transport promoter regions, inhibiting transcription. A similar negative regulator might be needed to trigger the copper switch to pMMO production.