Thailandamide, a Fatty Acid Synthesis Antibiotic That Is Coexpressed with a Resistant Target Gene

Abstract

Microbes encode many uncharacterized gene clusters that may produce antibiotics and other bioactive small molecules. Methods for activating these genes are needed to explore their biosynthetic potential. A transposon containing an inducible promoter was randomly inserted into the genome of the soil bacterium Burkholderia thailandensis to induce antibiotic expression. This screen identified the polyketide/nonribosomal peptide thailandamide as an antibiotic and discovered its regulator, AtsR. Mutants of Salmonella resistant to thailandamide had mutations in the accA gene for acetyl coenzyme A (acetyl-CoA) carboxylase, which is one of the first enzymes in the fatty acid synthesis pathway. A second copy of accA in the thailandamide synthesis gene cluster keeps B. thailandensis resistant to its own antibiotic. These genetic techniques will likely be powerful tools for discovering other unusual antibiotics.

Keywords: Burkholderia thailandensis; acetyl-CoA carboxylase; antimicrobial; global regulator; polyketide.

FIG 1

Inhibition of Salmonella by B. thailandensis atsR mutants requires the thailandamide synthesis genes. Saturated liquid cultures diluted out to produce single colonies (A) or 5 μl of saturated liquid cultures spotted on plates (B, C, and D) were grown for 24 h. A 200-fold dilution of a saturated culture of wild-type Salmonella was sprayed onto the surface, and these plates were grown overnight. A white bar representing a 5-mm width is included in each panel.

FIG 2

Transposon insertions in the thailandamide synthesis cluster and other loci disrupt inhibition by Burkholderia. Transposon insertions (gray triangles with black borders) at the tha (A), pgl (B), mnmE (C), BTH_I0093 (D), ast (E), and BTH_II2286 (F) loci disrupted inhibition of Salmonella. In panel A, thailandamide biosynthesis genes are in blue, and the acetyl-CoA carboxylase gene accA-2 is in green. The functions of proteins, as determined by Pfam (50) and InterPro (51) searches, are listed beneath genes. These proteins include an acyltransferase (AT), a pyruvate oxidoreductase (POR), a glutathione S-transferase (GST), a major facilitator superfamily (MFS) transporter, and helix-turn-helix (HTH) DNA-binding proteins.

FIG 3

Thailandamide isolated from wild-type and atsR mutants. (A) The structures of thailandamide A (12) and thailandamide B (33) are displayed. (B) HPLC traces from single plate extracts for three independent cultures of the wild-type strain or atsR::FRT mutant are shown. The agar was soaked in methanol, and the liquid was ethyl acetate extracted before being analyzed on the HPLC column. The baselines for the traces are offset on the y axis by different amounts to better show the differences between traces. The traces are also slightly offset on the x axis to better align the main peak. (C) An HPLC trace is displayed for compound that was HPLC-purified using solvent containing TFA.

FIG 4

Purified thailandamide kills Salmonella in liquid cultures. Approximately 4 × 10⁴ cells of stationary-phase Salmonella were added in a 200-μl volume to each well in a 96-well plate. Thailandamide B was added in 1 μl DMSO. The plate was incubated in the dark at 37°C with shaking. A sample was removed from each well at each time point, diluted appropriately, and spread on solid medium to determine the number of remaining viable cells.

FIG 5

Mutated residues in acetyl-CoA carboxylase (ACC) confer resistance to thailandamide in Salmonella. (A) The ACC enzyme transfers a CO₂ group (in bold) from biotin to the acetyl group in acetyl-CoA. ACC interacts with biotin when biotin is tethered to a lysine residue on biotin carboxyl carrier protein (BCCP). (B) The crystal structure of the E. coli ACC (35) is displayed. To show the likely binding sites for biotin and acetyl-CoA, PyMol was used to align the crystal structures of E. coli ACC (35) and Streptomyces coelicolor propionyl-CoA carboxylase (52). The S. coelicolor propionyl-CoA carboxylase was crystalized with biotin (blue) and propionyl-CoA (green). Biotin partly dips beneath the surface of the E. coli structure, which is probably due to small differences between the two structures. The residues in the Salmonella ACC that confer resistance to thailandamide are marked in red. The AccA half of the enzyme is displayed in white, and the AccD half is shown in gray. (C) The E. coli ACC structure (35) is displayed at a different angle without ligands. A stick representation of residues near the three mutated positions is also shown in the same orientation.

FIG 6

Burkholderia's second copy of AccA provides resistance to thailandamide in Burkholderia and Salmonella. Saturated cultures diluted 1:100 (A and B) or 1:200 (C) were spread or pipetted onto plates. Filter paper containing 8 nmol (A and C) or 20 nmol (B) of purified thailandamide B was placed onto each plate. The plates were incubated overnight.

FIG 7

Thailandamide inhibits pathogenic isolates of E. coli, P. aeruginosa, and S. aureus. Saturated cultures diluted 1:200 were spread onto plates. Filter paper containing 8 nmol of purified thailandamide B was placed onto each plate. The plates were incubated overnight.