EP2094851A2 - Voie de biosynthese et genes necessaires a la biosynthese d'acide tropodithietique dans silicibacter tm1040 - Google Patents

Voie de biosynthese et genes necessaires a la biosynthese d'acide tropodithietique dans silicibacter tm1040

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EP2094851A2
EP2094851A2 EP07868871A EP07868871A EP2094851A2 EP 2094851 A2 EP2094851 A2 EP 2094851A2 EP 07868871 A EP07868871 A EP 07868871A EP 07868871 A EP07868871 A EP 07868871A EP 2094851 A2 EP2094851 A2 EP 2094851A2
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tda
acid
tropodithietic
roseobacter
nucleic acid
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Robert Belas
Haifeng Geng
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The University of Maryland Biotechnology Institute
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
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    • C12P11/00—Preparation of sulfur-containing organic compounds
    • C—CHEMISTRY; METALLURGY
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms

Definitions

  • This invention relates to Roseobacter bacteria and to the production of antibiotics by use of such microbial species.
  • Roseobacters Bacteria of the Roseobacter clade of marine alpha-Proteobacteria stand out as some of the most critical players in the oceanic sulfur cycle due to the ability of several genera to degrade dimethylsulfoniopropionate (DMSP). While roseobacters are wide-spread throughout the marine ecosystem, their abundance is significantly correlated with DMSP-producing algae, especially prymnesiophytes and dinoflagellates, such as Prorocentrum, Alexandrium and Pfiesteria species. [0005] Roseobacters have abundant and diverse transporters, complex regulatory systems, multiple pathways for acquiring carbon and energy in seawater, with the potential to produce secondary, biologically active metabolites.
  • DMSP dimethylsulfoniopropionate
  • the present invention relates to Roseobacter bacteria and to the production of antibiotic tropodithietic acid (TDA) by use of such microbial species.
  • TDA antibiotic tropodithietic acid
  • the invention relates to an isolated nucleic acid encoding a megaplasmid
  • Another aspect of the invention relates to a protein encoded by a nucleic acid sequence represented by SEQ. ID. 1; wherein the protein is involved in the biosynthesis of tropodithietic acid by Roseobacter bacteria.
  • Yet another aspect of the invention relates to a protein encoded by a nucleic acid sequence represented by SEQ. ID. 2; wherein the protein is involved in the biosynthesis of tropodithietic acid by Roseobacter bacteria.
  • the invention relates to a protein encoded by a nucleic acid sequence represented by SEQ. ID. 3; wherein the protein is involved in the biosynthesis of tropodithietic acid by Roseobacter bacteria.
  • An additional aspect of the invention relates to a protein encoded by a nucleic acid sequence represented by SEQ. ID. 4; wherein the protein is involved in the biosynthesis of tropodithietic acid by Roseobacter bacteria.
  • Still another aspect of the invention relates to a method for producing an antibacterial composition comprising tropodithietic acid, the method comprising:
  • a further aspect of the invention relates to a method of treating or preventing bacterial disease in a subject in need of such treatment or prevention, comprising administering to said subject an antibacterial composition comprising tropodithietic acid isolated from bacteria of the
  • Yet another aspect of the invention relates to a plasmid pSTM3.
  • Another aspect of the invention relates to a compound selected from the group consisting of:
  • FIG. 1 When grown in static liquid media, Silicibacter sp. strain TM 1040 produces a yellow-brown pigment and has a large amount of antibacterial activity, which was measured by a well diffusion assay using V. anguillarum as the target organism (Materials and Methods). In contrast, pigment and antibacterial compound is very low under 30 0 C shaking conditions.
  • FIG. 2. Tropodithietic acid. Cl 8 reverse phase HPLC chromatograms of ethyl acetate extracts from TM 1040 and Phaeobacter 27-4. Insets show the UV spectra of the HPLC peak corresponding to the antibiotic activity. For 27-4, the peak is TDA.
  • FIG. 3 Genes required for synthesis of TDA in TM1040.
  • the black boxes indicate the ORF interrupted by the transposon. Arrows indicate ORFs transcriptional orientations and hatch marks indicate a break in the region.
  • Sulfur assimilation genes, tdaH, malY, cysl are located in the TM1040 chromosome. Phenylacetate catabolism genes are in the megaplasmid pSTMl.
  • B td ⁇ A-td ⁇ F genes reside on a cryptic plasmid, with their closest homologues found on the chromosome of P. denitrific ⁇ ns PD 1222.
  • td ⁇ H encodes sulfite oxidase domain protein
  • hik2 encodes two-component hybrid sensor and regulator
  • m ⁇ lY encodes ⁇ -C-S lyase (cystathionase); ⁇ snC encodes transcriptional regulator AsnC family
  • cysG encodes siroheme synthesis; hypo encodes hypothetical protein
  • cysl encodes sulfite reductase beta (siroheme- dependent);
  • cysH encodes adenylylsulfate reductase
  • gntR encodes GntR family transcriptional regulator
  • p ⁇ G, p ⁇ H, p ⁇ l, p ⁇ J, p ⁇ K encode respectively phenylacetic acid degradation protein complex protein 1, 2,3,4,5 ;td ⁇ A encodes LysR substrate binding domain protein; td ⁇ B encodes ⁇ -etherase; glutathione-S-transfera
  • P. denitrific ⁇ ns PD 1222 genome contains two chromosomes and one plasmid, whereas td ⁇ AB, td ⁇ CDE and td ⁇ F homologue genes located discretely in a 19 kb region of chromosome 1.
  • FIG. 4 Growth and TDA synthesis is affected by mutations in cysl.
  • TM1040 inverted triangles
  • the cysl mutant HG1220; circles
  • methionine closed symbols
  • methionine open symbols
  • growth was measured optically at 600 nm.
  • the Cysl " mutant cannot grow methionine, but does utilize cysteine.
  • Measurement of antibiotic activity indicates that the cysl defect also affects TDA synthesis, which is corrected by the addition of cysteine to the medium, but not methionine, DMSP, sulfite, or sulfate addition (table).
  • FIG. 5 shows that the cysl defect also affects TDA synthesis, which is corrected by the addition of cysteine to the medium, but not methionine, DMSP, sulfite, or sulfate addition (table).
  • TM1040 tda genes reside on a cryptic plasmid that undergoes a low frequency spontaneous loss.
  • A Pigment synthesis.
  • TM1040 (wt) produces a yellow-brown extracellular pigment that is correlated with TDA synthesis.
  • a tdaE:Tn mutant strain HG1265
  • a spontaneous mutant sm; TM1040SM
  • B Spontaneous loss of pigment and antibiotic activity results from a loss of tda genes.
  • PCR amplification of tdaE results in a band from wt and tdaE:Tn DNA, respectively, with the additional 2 kb in size of the tdaE:Tn product resulting from insertion of the transposon.
  • No product was amplified from the spontaneous nonpigment mutant (sm).
  • C PFGE separation of total DNA obtained from TM1040 (wt), the spontaneous nonpigmented mutant (sm), and the tdaE:Tn mutant. All three strains show a fuzzy band at ca. 130 kb, with a slight increases in the width of the wt and tdaE bands.
  • D Southern blot hybridization of the PFGE gel to labeled tdaE DNA.
  • the tdaE probe hybridized to the band migrating at ca. 130 kb in both wt and tdaE:Tn (first and third lanes), but failed to hybrid to the DNA obtained from the spontaneous nonpigmented mutant.
  • E Ncol digestion of plasmid DNA isolated from TM 1040 (wt), the spontaneous nonpigmented mutant (sm), and HG 1265 (tdaE:Tn), respectively. The digested DNAs were separated by electrophoresis and the band patterns compared to each other and to an in silico Ncol digestion of pSTM2 (supplemental data).
  • Total DNA was extracted from 13 roseobacters, TM1040, and a non-roseobacter control species (V. anguillarum), and used in a slot blot hybridization with labeled tda DNA. Positive hybridization was strongly correlated with measurable antibiotic activity (indicated by an asterick).
  • the strains used were: ISM: Roseovarius strain ISM; TM1038: Roseobacter sp. strain TM1038; TM1039, Roseobacter sp.
  • FIG. 8 The presence and relative abundance of each of the Tda proteins identified in TM 1040 (rows) in the GOS metagenomic database (via the internet website at hypertext transfer protocol address, camera.calit2.net/). The relative abundance is based on the total BLASTP matching sequences in the individual filters using a cutoff E value of 1E-20 (48).
  • the distribution of Tda proteins harbored on pSTM3 (TdaA-F) in the sample is remarkably different from the distribution of Paa and sulfur metabolism proteins (Cysl, MaIY, and TdaH), which have a more even distribution throughout the series of samples. Relative abundance is indicated by the size of the circle.
  • GOS sample numbers are indicated on the horizontal axis.
  • FIG. 9 A putative model of the TDA biosynthetic pathway based on the present genetic analysis.
  • the suggested pathway involved phenylacetate derivation from shikimate-chorismate and degradation pathway providing precursors (step 1-6) and an core oxidative ring-expansion pathway forming the seven carbon tropolone skeleton (step7 ⁇ 10) followed by sulfur-oxygen exchange (stepll ⁇ 15), consistent with the proposed TDA synthesis based on chemical labeling studies in Pseudomonas CB-104 (14).
  • the protein assignment was based on predicted functions.
  • the present invention relates to Roseobacter bacteria and to the production of tropodithietic acid (TDA) by use of such microbial species.
  • TDA tropodithietic acid
  • the symbiotic association between the roseobacter Silicibacter sp. TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment.
  • the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA).
  • tdaA-F six of the genes, referred to as tdaA-F, could not be found on the annotated TM1040 genome and were instead located on a previously unidentified cryptic megaplasmid (ca. 130 kB; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp.
  • TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA:DNA hybridization in 88 % of a diverse collection of 9 roseobacters with known antibiotic activity.
  • roseobacters employ a common pathway for TDA biosynthesis that involves plasmid-encoded proteins.
  • metagenomic library databases and a bioinformatics approach a pronounced difference in the biogeographical distribution between the critical TDA synthesis genes was observed, implying substantial environmental preference differences among these genes.
  • the present invention in another specific aspect relates to the interaction of a roseobacter, Silicibacter sp. TM1040, and Pfiesteria piscicida.
  • Silicibacter sp. TM1040 (hereafter referred to as TM1040) is isolatable from laboratory microcosm culture of heterotrophic DMSP-producing dinoflagellate P. piscicida.
  • Marine algae are major producers of DMSP in the marine environment while members of the Roseobacter clade are capable of DMSP catabolism.
  • TM 1040 degrades DMSP via a demethylation pathway producing 3-methylmercaptopropionate (MMPA) as a major breakdown product.
  • MMPA 3-methylmercaptopropionate
  • the bacteria respond via chemotaxis to dinoflagellate homogenates, and are specifically attracted to DMSP, methionine, and valine.
  • TM1040 motility is important in the initial phases of the symbiosis. Once the bacteria are in close proximity to their host, TM 1040 forms a biofilm on the surface of the dinoflagellate.
  • the symbiosis includes two parts: one that involves chemotaxis and motility, and a second step in which a biofilm predominates.
  • TM1040 consists of a 3.2 Mb chromosome and two plasmids, pSTMl (823 Kb) and pSTM2 (131 Kb) (41).
  • Tropolone ring 2 is a seven member aromatic tropolone ring, which is highly significant as tropolone derivatives, notably hydroxylated forms, are widely seen as medically important sources of antibacterial, antifungal, antiviral, and antiparasitic agents.
  • Thiotropocin another tropothione derivative closely related to TDA, can be synthesized from shikimate by an oxidative ring expansion of phenylacetic acid.
  • a marine basal minimal medium (MBM; per liter: 8.47g Tris HCl, 0.37 g of NH 4 Cl, 0.0022 g of K 2 HPO 4 , 11.6 g NaCl, 6 g MgSO 4 , 0.75 g KCl, 1.47 g CaCl 2 -2H 2 O, 2.5 mg FeEDTA; pH 7.6, 1 ml of RPMI- 1640 vitamins [Sigma R7256]) was used for determining carbon and sulfur requirements. Sole carbon sources were added at a final concentration of 1 g/1.
  • Escherichia coli strains were grown in Luria-Bertani (LB) broth or on LB agar containing 1.5% Bacto Agar (Becton Dickinson, Franklin Lakes, N.J.). As appropriate, kanamycin was used at 120 ⁇ g per ml for Roseobacter strains and 50 ⁇ g per ml for E. coli. [0037] Characterization of antibiotic
  • Tda is defined as a strain lacking a detectable zone of clearing on V. anguillarum.
  • Strains determined to be Tda- by the modified well-diffusion assay were further tested by incubation at 3O 0 C for 48 h in 2216 marine broth without shaking. Bacteria were removed by filtering through a 0.22 ⁇ m MCE membrane (Millex ,Millipore, Bedford, MA) and the antibacterial activity of the supernatant measured using the V. anguillarum well diffusion assay, as described by Bruhn et al. [0041] Sole carbon and sulfur source growth
  • Chromosomal DNA was extracted from bacterial cells by routine methods or by the DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA). Plasmid DNA was prepared by the alkaline lysis method, digested with Ncol (New England Biolabs, Beverly, MA), and the resulting restriction fragments were separated by agarose gel electrophoresis in Tris-acetate- EDTA (TAE) buffer.
  • TAE Tris-acetate- EDTA
  • Pulsed Field Gel Electrophoresis was performed using a CHEF DR-III clamped homogeneous electric field system (Bio-Rad, Richmond, Calif.) with a 1% agarose gel, a 3- to 15-s pulse ramp, an electrophoresis rate of 6.0 V/cm with an included angle of 120° at a constant temperature of 14 0 C, and a run time of 26 h.
  • Gels were stained with ethidium bromide (EB) and visualized with a Typhoon 9410 (Amersham Biosciences, Piscataway, NJ. ).
  • Standard PCR amplification conditions were 100 ⁇ M dNTP each, 0.2 ⁇ M of each primer, 1 U Taq DNA polymerase (New England Biolabs, Beverly, MA) in Ix reaction buffer (New England BioLabs) with an initial denaturing step at 94 0 C for 3 min, followed by 30 cycles of 94 0 C for 1 min each, annealing at 55 0 C for 30 s, and an elongation at 72 0 C for 1 min.
  • PCR amplification was conducted with a forward primer complementary to tdaA (5 ' -CGCTTTCCGGAACTGGAGAT-S') and a reverse primer complementary to tdaE (5 ' -GGCTGCCGTATAGTTTCAGCA-S') using the. Expand Long Template PCR System (Roche Applied Science, Indianapolis, IN) and the PCR program conditions and cycle parameters as described by the supplier. [0053] DNA hybridization [0054] DNA:DNA hybridization by Southern 'slot' blot (3) was used to detect the presence of tda genes in other roseobacters.
  • the roseobacter strains used were: Phaeobacter strain 27-4, Roseobacter algicola ATCC 51442, Roseobacter denitrificans ATCC 33942, Roseobacter litoralis ATCC 49566, Roseobacter sp. strain TM1038, Roseobacter sp. strain TM1039, Roseovarius sp. strain TM1035, Roseovarius sp. strain TM1042, Roseovarius strain ISM, Silicibacter pomeroyi DSS-3, Silicibacter sp. strain TM1040, Sulfitobacter strain EE36, Sulfitobacter strain 1921, Sulfitobacter strain SE62, and Vibrio anguillarum 90-11-287.
  • the membrane was incubated at 25 0 C overnight with a double-stranded DNA probe prepared by Hind III digestion of a plasmid bearing tdaA cloned from strain HGl 310 that was labeled with digoxigenin-dUTP using random priming as recommended by the manufactures (Roche). Unbound labeled DNA was removed from the membrane by 2 x5 min in 2xSSC, 0.1% SDS followed by 2 xl5 min in 0.2xSSC, 0.1% SDS (3). In the southern blot, the membrane was prehybridized for 30 min in the same buffer to which was added a tdaD gene probe, and the probe allowed to hybridize overnight at 42°C. The blots were washed under high stringency conditions following the manufacturer's protocol (Roche applied science) and exposed to Lumi- film chemiluminescent detection film (Roche) for subsequent detection of the hybridization signal.
  • a double-stranded DNA probe prepared by Hind III digestion of a
  • TM 1040 produces the sulfur-containing antibiotic tropodithietic acid
  • TM1040 produces an extracellular broad spectrum antibacterial compound capable of inhibiting or killing many bacteria. We found that greater antibacterial activity occurred when the bacteria were grown in a nutrient broth culture under static conditions, i.e., no shaking, compared to shaking conditions (11 mm; Fig. 1). Under static conditions, TM1040 cells attached to one another forming rosettes and produced a very distinct yellow-brown pigment (Fig. 1). These phenotypes are consistent with Phaeobacter 27-4 and other roseobacters.
  • TM 1040 produces an antibiotic and shares common phenotypic traits with other roseobacters, notably Phaeobacter 27-4 whose antibiotic is tropodithietic acid (TDA). We therefore hypothesized that the antibacterial compound produced by TM1040 may also be tropodithietic acid.
  • TDA tropodithietic acid
  • Cell-free supernatants were collected independently from both TM 1040 and 27-4, ethyl acetate extraction of the supernatants was used to separate TDA from other compounds, and the concentrated extract was analyzed by HPLC.
  • Fig. 2 The resulting elution chromatograms and subsequent UV spectra of the putative peak of TDA from TM1040 and 27-4 are shown in Fig. 2. Both chromatograms and UV spectra are nearly identical, indicating chemically similar metabolites are produced by both strains. A compound with a retention time of 4.2 min (indicated in Fig. 2) is observed in both chromatograms and has been positively identified as TDA in 27-4.
  • the equivalent 'TDA peak' from TM1040 has a UV spectrum that overlaps with that of published spectrum of TDA obtained from 27-4, with four major absorptions at 210 nm, 304 nm, 355 nm and 452 nm.
  • the location of the transposon insertion site in each of the 81 Tda " mutants was determined by sequencing TM1040 DNA adjacent to the transposon. The pair of sequences (both sides of the transposon insertion point) obtained from each mutant was used to search the annotated TM 1040 genome to identify the mutated gene. Surprisingly, we were unable to find homologs in the genome for 32 or nearly 40% of the Tda " mutants, yet these DNAs overlapped permitting assembly into one large contiguous DNA fragment of 4.5 kb harboring at least 6 ORFs that we have called tdaA-F (Table 2 and Fig. 3A).
  • tdaA-F represent DNA that is not part of the original annotation of the genome, suggesting that this DNA may have been lost from the sequenced variant of TM1040.
  • TDA biosynthesis genes resided on a 130 kb cryptic plasmid.
  • Forty nine Tda " mutants had transposon insertions in genes found in one of the three DNAs that make up the genome. Due to the observation of a low frequency spontaneous loss of TDA synthesis and knowledge of the existence of tdaA-F, we analyzed each of the 49 genomic Tda " strains for the presence of tdaA-F.
  • paaGHIJK encodes a ring- hydroxylating complex of proteins that is responsible for the first step in the aerobic catabolism of phenylacetate involving Coenzyme A (CoA) activation, producing 1, 2-dihydro- phenylacetate-CoA.
  • CoA Coenzyme A
  • the finding that mutations in paa genes affects TDA synthesis is consistent with the biochemical evidence of phenylacetate metabolism in thiotropocin synthesis.
  • Mutants with defects in phenylacetate metabolism were also unable to grow on phenylalanine, phenylacetic acid, tryptophan, sodium phenylpyruvate or phenylbutyrate as a sole carbon source (Table 3).
  • TDA is a disulfide-modified tropolone compound, indicating that sulfur metabolism must be involved in TDA synthesis.
  • This hypothesis is supported by the identification of 3 Tda " mutants (Table 2) each with a transposon inserted in a gene whose product is involved in sulfur metabolism: cysl, malY, and an ORF (tdaH) with homology to sulfite oxidase (Table 2).
  • the identification of these genes suggests that sulfur from reductive sulfur pathways is used and incorporated into TDA, which was tested by observing growth of the sulfur-metabolism mutants on a minimal medium containing a sole sulfur source (Materials and Methods). The results are shown in Fig. 4.
  • the cysl mutant grew when provided complex sulfur sources or cysteine and was unable to utilize DMSP, SO 3 2" , SO 4 2" , or methionine.
  • the addition of cysteine to the medium resulted in enhanced growth of the cysl mutant as well as increased synthesis of TDA (Fig. 4).
  • TDA biosynthesis genes resided on a 130 kb cryptic plasmid
  • tdaA-F genes were not part of the annotated TM1040 genome and were absent in spontaneous Tda " mutants.
  • these genes share their strongest homology with a similar set of genes in Paracoccus denitrificans PD1222 chromosome 1 (Accession number: NC_008686), a non-motile alphaproteobacterium first isolated from soil by Beijerinck. As shown in Fig.
  • TdaA (Table 2) has homology with LysR regulatory proteins, possessing a helix-turn-helix and a LysR substrate-binding domain (57). TdaA is the only regulatory protein uncovered in this study, perhaps indicating that it is the sole regulator of TDA synthesis. The remaining ORFs encode putative enzymes.
  • TdaB contains a glutathione S- transferase (GST) domain and belongs to the bacterial GST protein family (Table 1).
  • TdaC has an amino acid domain with homology to prephenate dehydratase (PheA), an enzyme involved in the conversion of chorismate to prephenate, a step in the pathway leading to phenylacetate synthesis.
  • PheA prephenate dehydratase
  • Table 1 Bacterial strains and plasmids used.
  • TdaD is anticipated to be a member of the thioesterase superfamily of acyl-CoA thioesterases (Table 2)
  • TdaE encodes a putative acyl-CoA dehydrogenase (ACAD)
  • ACAD putative acyl-CoA dehydrogenase
  • TdaF has homology to aldehyde dehydrogenase.
  • TM1040 a spontaneous Tda " nonpigmented strain of TM1040 (TM1040SM), and HG1265 (tdaE::Tn)
  • TM1040SM a spontaneous Tda " nonpigmented strain of TM1040
  • HG1265 tdaE::Tn
  • TM1040, TM1040SM, and HG1265 we isolated plasmids from each of the three strains (TM1040, TM1040SM, and HG1265) and subjected each mixture to Ncol digestion (Fig. 5E), chosen because an in silico Ncol digestion of pSTM2 provided a recognizable pattern of DNA fragments. As shown in Fig. 5E, the TM1040SM DNA digest had much fewer bands than wild- type DNA or DNA from tdaE:Tn. This would be expected if the TM1040SM strain lost a large plasmid.
  • each plasmid was PCR positive for the tda genes (data not shown) and the set of four shared many common bands, they had remarkably different patterns indicating deletion and/or rearrangements had occurred during or after transfer of pSTM3 to E. coli.
  • the reason and molecular mechanism underlying these band pattern differences is not known; however, the sum of the results indicates that TM1040 harbors a ca. 130 kb plasmid, pSTM3, which is essential for TDA and pigment biosynthesis and which may be spontaneously lost in laboratory culture.
  • TM 1040 (Table 4), suggesting that these two roseobacters share a common TDA biosynthesis and regulation scheme.
  • the remaining 9 genes were not identified as important to TDA synthesis in TM1040 and had varying degrees of homology to genes in the annotated TM1040 genome, but, unlike TM1040, were not part of the phenylacetate or reductive sulfur pathways.
  • the one exception is 27-4 metF (Table 4), which may possibly be involved in sulfur metabolism.
  • JBB1030 EF139217 tdaA LysR substrate binding Paracoccus denitrificans domain protein PD 1222 regulatory protein,
  • antibiotic activity including antibacterial activity from roseobacters, e.g., a compound that produces a probiotic effect on scallop larvae and is antagonistic to ⁇ -Proteobacteria strains, as well as a compound that is antagonistic against fish larval bacterial pathogens. From our data, it is likely that much of the antibiotic activity seen in roseobacters is due to plasmid-borne tda genes that can be difficult to maintain in laboratory conditions.
  • 27-4 possesses at least two plasmids of ca. 60 kb and 70 kb respectively.
  • One or both of these plasmids may be involved in TDA biosynthesis of 27-4 and tdaA and tdaB, identified by transposon insertion mutagenesis in 27-4 Tda " mutants, reside on one of these plasmids.
  • Instability of pSTM3 is also apparent when the plasmid is transformed into a nonroseobacter host, e.g., E. coli. As shown in Fig.
  • TM1040 possesses varied capabilities to achieve horizontal gene transfer, including the presence of several prophage genomes in the bacterium's genome, one of which is homologous to the gene transfer agent of other alphaproteobacteria, and many of genes on pSTM2 are homologs of the vir system of Agrobacterium tumefaciens. The A.
  • tumefaciens Ti plasmid transferred by Vir Type IV secretion, requires RepABC, suggesting that a similar mechanism may allow pSTM3 transfer to other organisms.
  • Plasmids similar to pSTM3, such as pSymA of Sinorhizobium meliloti and the Ti plasmid, are important for the proper interaction of those bacteria and their respective hosts, and TM1040 pSTM3 and pSTM2 may correspondingly serve to enhance the TM1040- dinoflagellate symbiosis. [0088] It is important to note that TDA activity and biosynthesis depend on culture conditions and the physiology of TM1040.
  • TDA activity is significantly enhanced when TM1040 is cultured in a static nutrient broth, a condition that accentuates biofilm formation.
  • the symbiosis includes two phases: the motile phase in which TM 1040 cells actively respond to dinoflagellate- derived molecules by swimming towards the host, and sessile phase, whereupon having located the zoospore, the bacteria cease to be motile and form a biofilm on the surface of the dinoflagellate.
  • TDA Biosynthesis of TDA has several potentially beneficial effects on the TM1040- dinoflagellate symbiosis.
  • TDA is likely to benefit the dinoflagellate by acting as a probiotic with antibacterial activity whose action prevents the growth and colonization of bacteria on the surface of the dinoflagellate that could potentially harm the zoospore.
  • the antibacterial activity of TDA may enhance the growth of TM 1040 cells attached to the zoospore by warding off other biofilm-forming bacteria that compete with TM 1040 for space on the surface of and nutrients from P. piscicida.
  • DMSP appears not to be a primary source of the sulfur atoms of TDA.
  • One or more non-DMSP sulfur-containing metabolites produced by the dinoflagellate may be used by TM1040 in the biosynthesis of TDA.
  • Tda proteins are found in either the genomes of other sequenced roseobacters or in the CAMERA metagenomic library (Fig. 8).
  • Tda proteins were not found. For example, amino acid sequence divergence between Tda proteins of TM1040 and other roseobacters could result in BLASTP E values greater than our chosen cutoff (1E-20). This argument may also be applied to the metagenomics search.
  • TdaB a homolog of glutathione S-transferase, is a potential agent in the addition of sulfur to the nascent TDA molecule.
  • the compounds shown in Fig. 9 include the following: Table 5
  • LysR homolog in TdaA is consistent with the regulation of TDA biosynthesis involving a cofactor.
  • LysR cofactors can function as precursor molecules required to synthesize the final product, implicating molecules in the shikimate pathway, phenylacetate, or other TDA precursors as being required for maximal expression of the tda genes. Consistent therewith, modifications of the broth by addition of phenylalanine and histidine significantly increase production of TDA from Phaeobacter T5.
  • TDA TDA synthesis by roseobacters
  • pSTM3 previously unknown megaplasmid
  • the backbone of TDA is a seven member aromatic tropolone ring, which is highly significant as tropolone derivatives, notably hydroxylated forms, are medically important sources of antibacterial, antifungal, antiviral, and antiparasitic agents.
  • tropolone and derivatives can be difficult, making natural sources of tropolone precursors often the preferred choice as starting material for the synthesis of new tropolone antibiotics.
  • the mutants obtained in this study may lead to the development of bacterial sources of medically important tropolone compounds and a suite of new antimicrobial agents based on TDA.
  • CAATGCTGACAGGGGTATTCGGGATGCATCGGCGCAGGAATGCGCGATTCCCAGAC CAGACCGGCTCAGGTCGGGGTGGTTGTAGGTTTTGCCCAACGCCTCCACGGCGGTCT GTGGCCGCCAGAGCGCGTAGCTGTACTTGGCGTTGAACCCGGCCACCAACGCATCGC TCAGGGCGACATTCAACACGCCACATGCGCGTTTCCTCGAGAAGAGGCAGCGCGCC AGGCCGCGCAGAGAG
  • TCTTCAGGATCGATCTTCGGGTTCATACC repC GAATTCCTCGGCGCGGACATCCTCTGCCAGCATTTCGATCTCTTGCGCACGCAGCCG
  • Another aspect of the invention relates to a methodology for purification of TDA and intermediate compounds, including the use of solid phase extraction techniques to obtain tropodithietic acid from Silicibacter sp. TM1040.
  • a still further aspect of the invention relates to a method of purification of TDA by
  • Roseobacter 27-4 was grown in 500 ml MB in a 5 liter volumetric flask at 25 0 C for 4 days. 2. The cells were removed by centrifugation (10,000 x g for 10 min).
  • the invention provides an effective and useful biosynthetic capability for the production of tropodithietic acid (TDA) by use of Roseobacter bacteria.
  • TDA is a useful sulfur- containing antibiotic compound.
  • the biosynthetic route of the present invention enables scalable production of TDA and TDA derivatives.

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Abstract

L'association symbiotique entre le roséobacter Silicibacter sp. TM1040 et le dinoflagellé Pfiesteria piscicida entraîne une chimiotaxie bactérienne impliquant le diméthylsulfoniopropionate (DMSP) produit par le dinoflagellé, une méthylation de DMSP et, enfin, la formation d'un film biologique sur la surface du récepteur. La formation de ce film biologique coïncide avec la production d'un antibiotique et d'un pigment jaune-brun. Cet antibiotique est un composé contenant du soufre, acide tropodithiétique (TDA). La mutagénèse sélective par insertion de transposons a permis d'identifier 12 gènes jouant un rôle important dans la biosynthèse de TDA par la bactérie et toute mutation dans ces gènes a pour effet une perte de l'activité antibiotique (Tda) et de la production de pigment. L'invention concerne une voie de biosynthèse et de régulation pour la biosynthèse de TDA dans des roséobacters.
EP07868871A 2006-11-27 2007-11-27 Voie de biosynthese et genes necessaires a la biosynthese d'acide tropodithietique dans silicibacter tm1040 Withdrawn EP2094851A2 (fr)

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