Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills

Abstract

Shipworms are marine wood-boring bivalve mollusks (family Teredinidae) that harbor a community of closely related Gammaproteobacteria as intracellular endosymbionts in their gills. These symbionts have been proposed to assist the shipworm host in cellulose digestion and have been shown to play a role in nitrogen fixation. The genome of one strain of Teredinibacter turnerae, the first shipworm symbiont to be cultivated, was sequenced, revealing potential as a rich source of polyketides and nonribosomal peptides. Bioassay-guided fractionation led to the isolation and identification of two macrodioloide polyketides belonging to the tartrolon class. Both compounds were found to possess antibacterial properties, and the major compound was found to inhibit other shipworm symbiont strains and various pathogenic bacteria. The gene cluster responsible for the synthesis of these compounds was identified and characterized, and the ketosynthase domains were analyzed phylogenetically. Reverse-transcription PCR in addition to liquid chromatography and high-resolution mass spectrometry and tandem mass spectrometry revealed the transcription of these genes and the presence of the compounds in the shipworm, suggesting that the gene cluster is expressed in vivo and that the compounds may fulfill a specific function for the shipworm host. This study reports tartrolon polyketides from a shipworm symbiont and unveils the biosynthetic gene cluster of a member of this class of compounds, which might reveal the mechanism by which these bioactive metabolites are biosynthesized.

Fig. 1.

Chemical structure of tartrolons and structurally related compounds.

Fig. 2.

HR-MS and tandem MS spectra of the crude extracts of each of T. turnerae wild type (Upper) and region 2 mutant, AH02 (Lower). The high-resolution signal corresponding to compound 2 is present in the wild type but absent from AH02. The high-resolution mass of the main peak in the wild-type spectrum is 873.4174; this mass is absent in AH02. The mass of the main peak in the mutant AH02 is 873.5529, a mass which also is present in the wild type as a minor compound, In addition, the MS/MS fragmentation patterns of these peaks were different, confirming the absence of compound 2 in the disrupted mutant.

Fig. 3.

Biosynthesis scheme of tartrolons from the trt cluster. Compounds 1 and 2 are color coded based on the corresponding domains. Plasmid pDMrg2KS was used to disrupt KS1. ACP, acyl carrier protein; AT, acyltransferase; cm^r, chloramphenicol resistance gene; DH, dehydratase; GNAT, Gcn 5-N-acetyl transferase; KR, ketoreductase; KS, ketosynthase; M, module; MT, methyl transferase; PKS, polyketide synthase; R, enoyl reductase; TE, thioesterase.

Fig. 4.

ML-reconstituted tree of full-length unedited KS domains from trans-AT PKS enzymes. For clarity, known clades and clades not relevant to TrtKS were collapsed. The KS domains are numbered according to the occurrence in the gene cluster starting from the 5′ end, as previously established (20). The numbers in parenthesis indicate total number of KS domains in the clade, number of KS with known function and matching specificity/number of KS with unknown function/number of KS with known function and mismatching specificity, exactly as previously described (21). Alb, albicidin; Bae, bacillaene (Bacillus amyloliquefaciens); Bry and BryX, bryostatin; BBR and BBR2, Brevibacillus brevis NBRC 100599 clusters BBR47_31930-32020 and BBR47_39780-39920; BCER, Bacillus cereus BSGC 6E1 cluster; BT2, Burkholderia thailandensis MSMB43 cluster; Bat, batumin; BATR, Bacillus atrophaeus 1942 cluster; BTP, Bacillus thuringiensis pondicheriensis BGSC 4BA1 cluster; CACI, Catenulispora acidiphila DSM 44928; CC, CC2, and CC3, Clostridium cellulolyticum H10 clusters Ccel_0858-0868, Ccel_2373-2386, and Ccel_0965-0980; Chi, chivosazol; Cor, corallopyronin; Dif, difficidin; Dsz, disorazol; Els, elansolid; Etn, etnangien; GU, Geobacter uraniireducens Rf4; Kir, kirromycin; Lkc, lankacidin; Lnm, leinamycin; Mgs, migrastatin; MICAU, Micromonospora aurantiaca ATCC 27029 cluster; Mmp, mupirocin; Mln, macrolactin; MSP, Micromonospora sp. ATCC 39149; Onn, onnamide; Ozm, oxazolomycin; Ped, pederin; Pel, Peltigera membranaceae cluster; PPA, Plesiocystis pacifica SIR-1 cluster; Psy, psymberin; Rhi, rhizoxin; SBI, Streptomyces bingchenggensis BCW-1 cluster; Sor, sorangicin; SG, Streptomyces griseus NPRC 13350 cluster; Ta, myxovirescin; Tai, thailandamide; Trt, tartrolon (T. turnerae T7901); Vir, virginiamycin M.

Fig. 5.

qPCR of trtF amplified from T. turnerae T7901 SBM broth cultures, the same medium under low inorganic phosphate (low Pi), and iron-starved (low Fe) conditions. Expression values were normalized to ftsZ.

Fig. 6.

In vivo expression analysis of the trt cluster in shipworms. (A) Reverse-transcriptase PCR products with Lyrodus pedicellatus gills total RNA and primer pairs specific for the trtD, trtE, and trtF PKS-coding genes and for the constitutively expressed ftsZ as positive control (Left). Control PCR reactions with Taq polymerase, primer pairs for ftsZ, and Lyrodus pedicellatus gills total RNA or DNA (Right). (B) Analysis of trtD in vivo expression by qPCR using bulk RNA from three different L. pedicellatus shipworms.

Fig. 7.

HPLC, HR-MS, and MS/MS analysis of shipworm crude extract. HPLC chromatogram of a representative crude extract of a L. pedicellatus shipworm (Top), HR-MS (Middle), and MS/MS fragmentation pattern (Bottom) of the peak corresponding to that of compound 1 (Fig. S2).

Fig. 8.

Potential functions of tartrolon in the shipworm–microbial symbiosis. Tartrolon is proposed to participate in bacterial inhibition, either in the gills (Right) by inhibiting certain bacterial phylotypes or in the cecum (Left) by preventing microorganisms from scavenging glucose.

Fig. P1.

Proposed model for the antibacterial activity of tartrolon E (structure shown) in shipworm–microbial symbiosis. Tartrolon is proposed to inhibit microorganisms either in the gills (to inhibit competing symbionts) (Right) or in the cecum (to prevent microorganisms from scavenging glucose) (Left).