CA2670626A1 - Biosynthetic pathway and genes required for tropodithietic acid biosynthesis in silicibacter tm1040 - Google Patents

Abstract

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). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in loss of antibiotic activity (Tda ) and pigment production. A biosynthetic and regulatory pathway for TDA biosynthesis in roseobacters is described.

Description

BIOSYNTHETIC PATHWAY AND GENES REQUIRED FOR TROPODITHIETIC

GOVERNMENT RIGHTS IN INVENTION

[0001] Work related to the invention was conducted in the performance of National Science Foundation Grant MCB0446001. The United States Government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application 60/861,117 filed November 27, 2006 in the names of Robert Belas, et al. for "BIOSYNTHETIC
PATHWAY AND GENES REQUIRED FOR TROPODITHIETIC ACID BIOSYNTHESIS IN
SILICIBACTER TM1040." The disclosure of U.S. Provisional Patent Application 60/861,117 is hereby incorporated herein by reference in its entirety, for all purposes.

BACKGROUND OF THE INVENTION
Field of the Invention [0003] This invention relates to Roseobacter bacteria and to the production of antibiotics by use of such microbial species.

Description of the Related Art [0004] 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.

SUMMARY OF INVENTION

[0006] The present invention relates to Roseobacter bacteria and to the production of antibiotic tropodithietic acid (TDA) by use of such microbial species.

[0007] In one aspect, the invention relates to an isolated nucleic acid encoding a megaplasmid (pSTM3) of Silicibacter sp. TM1040, wherein the nucleic acid comprises genes involved in tropodithietic acid biosynthesis of Roseobacter bacteria.

[0008] 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.

[0009] 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.

[0010] In another aspect, 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.

[0011] 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.

[0012] A further aspect of the invention relates to an antibacterial composition comprising tropodithietic acid isolated from bacteria of the Roseobacter clade [0013] Another aspect of the invention relates to a method for producing an antibacterial composition comprising tropodithietic acid, the method comprising:

a) culturing Silicibacter sp. TM1040 in a culture medium supporting growth of the bacterium and production of tropodithietic acid; and b) separating the tropodithietic acid from the culture medium; and c) purifying the tropodithietic acid by high performance liquid chromatography.

[0014] Still another aspect of the invention relates to a method for producing an antibacterial composition comprising tropodithietic acid, the method comprising:
a) culturing Roseobacter sp.27-4 in a culture medium supporting growth of the bacterium and production of tropodithietic acid; and b) separating the tropodithietic acid from the culture medium; and c) purifying the tropodithietic acid by high performance liquid chromatography.

[0015] 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 Roseobacter clade.

[0016] Yet another aspect of the invention relates to a plasmid pSTM3.

[0017] Another aspect of the invention relates to a compound selected from the group consisting of:
1, 2-dihydro-phenylacetyl-CoA;
2-hydroxy-7-oxo-cyclohepta-3,5-dienecarboxylic acid;
2,7-; dihydroxy-cyclohepta-1,3,5-trienecarboxylic acid;
2,7-dihydroxy-3-oxo-cyclohepta-1,4,6-trienecarboxylic acid;
2,7-dihydroxy-3-thioxo-cyclohepta-1,4,6-trienecarboxylic acid; and 7-hydroxy-2-mercapto-3-thioxo-cyclohepta-1,4,6-trienecarboxylic acid.

[0018] Other aspects, features and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1. When grown in static liquid media, Silicibacter sp. strain TM1040 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 C shaking conditions.

[0020] FIG. 2. Tropodithietic acid. C18 reverse phase HPLC chromatograms of ethyl acetate extracts from TM1040 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.

[0021] 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. (A) Sulfur assimilation genes, tdaH, malY, cysl, are located in the TM1040 chromosome. Phenylacetate catabolism genes are in the megaplasmid pSTM1.
(B) tdaA-tdaF genes reside on a cryptic plasmid, with their closest homologues found on the chromosome of P. denitrificans PD1222. tdaH encodes sulfite oxidase domain protein; hik2 encodes two-component hybrid sensor and regulator; malY encodes (3-C-S lyase (cystathionase);
asnC 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; paaG, paaH, paa7, paaJ, paaK encode respectively phenylacetic acid degradation protein complex protein 1,2,3,4,5;tdaA encodes LysR substrate binding domain protein; tdaB
encodes (3-etherase;
glutathione-S-transferase; tdaC encodes prephenate dehydratase; tdaD, 4-hydroxybenzoyl-CoA
thioesterase; tdaE encodes Acyl-CoA dehydrogenase; tdaF encodes phosphopantothenoylcysteine decarboxylase. P. denitrificans PD1222 genome contains two chromosomes and one plasmid, whereas tdaAB, tdaCDE and tdaF homologue genes located discretely in a 19 kb region of chromosome 1.

[0022] FIG. 4. Growth and TDA synthesis is affected by mutations in cysl.
TM1040 (inverted triangles) and the cysl mutant (HG1220; circles) were grown in minimal medium containing either methionine (closed symbols) or methionine (open symbols), and growth was measured optically at 600 nm. Unlike the wild-type, the Cyst 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).

[0023] FIG. 5. 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. In contrast, a tdaE:Tn mutant (strain HG1265) and a spontaneous mutant (sm; TM1040SM) are nonpigmented and have lost the ability to produce both TDA and pigment. (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 TM1040 (wt), the spontaneous nonpigmented mutant (sm), and (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). The pattern of fragments from sm DNA matched the predicted pSTM2 Ncol digestion, while both wt and tdaE DNA patterns showed evidence of additional restriction fragments. (F).
Southern blot hybridization of Ncol-digested plasmid DNA to tdaE. A tdaE probe hybridizes to one fragment in wt and tdaE:Tn DNA cut with Ncol, but to any fragments produced from Ncol digestion of plasmid. The increase in the size of the fragment in tdaE:Tn results from the insertion of the 2 kb transposon.

[0024] FIG. 6. Ncol digestion patterns of pSTM3 transformed into E. coli. A
mixture of plasmid pSTM3-1265 (pSTM3 harboring a transposon in tdaE) and pSTM2 was isolated from and the DNAs used to transform E. coli (Materials and Methods). Each of the plasmids harbored in the resulting Kanr transformants was purified, digested with Ncol, and the resulting DNA
fragments separated by agarose gel electrophoresis. Of the total DNAs examined, four types of band patterns emerged and are shown in lanes 1-4, respectively.

[0025] FIG. 7. DNA from other roseobacter species hybridizes to tda DNA. 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. strain TM1039; 33942, Roseobacter denitrificans ATCC 33942;
SE62, Sulfitobacter strain SE62; 49566, Roseobacter litoralis ATCC 49566; DSS-3, Silicibacter pomeroyi DSS-3; EE36, Sulfitobacter strain EE36; 1921, Sulfitobacter strain 1921; TM1040, Silicibacter sp. TM1040; V. a, Vibrio anguillarum; 51442, Roseobacter algicola ATCC 51442;
27-4, Phaeobacter 27-4; TM1035, Roseovarius sp. strain TM1035; and, TM1042, Roseovarius sp. strain TM1042.

[0026] FIG. 8. The presence and relative abundance of each of the Tda proteins identified in TM1040 (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, Ma1Y, 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.

[0027] 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 (step11-15), consistent with the proposed TDA synthesis based on chemical labeling studies in Pseudomonas CB-104 (14). The protein assignment was based on predicted functions.
DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to Roseobacter bacteria and to the production of tropodithietic acid (TDA) by use of such microbial species.

[0029] 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). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in loss of antibiotic activity (Tda ) and pigment production. Unexpectedly, 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.
These data suggest that roseobacters employ a common pathway for TDA
biosynthesis that involves plasmid-encoded proteins. Using 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.

[0030] 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.
TM1040 degrades DMSP via a demethylation pathway producing 3-methylmercaptopropionate (MMPA) as a major breakdown product. 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, TM1040 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.

[0031] Specific phenotypes, e.g., the ability to produce antibacterial compounds and biofilm formation, may give members of the Roseobacter clade a selective advantage, and help to explain the dominance of members of this clade in association with marine algae. The production of an antibiotic activity is observed in roseobacters and is hypothesized to provide an advantage when colonizing phytoplanktonic hosts, such as dinoflagellates. The genome of TM1040 consists of a 3.2 Mb chromosome and two plasmids, pSTM1 (823 Kb) and pSTM2 (131 Kb) (41). A comparison between TM1040 and two other roseobacters (Silicibacter pomeroyi DSS-3 and Jannaschia sp. CSS-1) suggests that 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.

[0032] Biologically active metabolites, including antibacterial compounds, are obtainable from roseobacters. A sulfur-containing antibiotic compound, tropodithietic acid (TDA), has been isolated and chemically characterized from Phaeobacter 27-4, hereafter called 27-4, and Roseobacter T5. The chemical backbone of TDA (shown in Fig. 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.

[0033] We have used both genomic and genetic techniques to identify the genes and proteins required for TDA synthesis in TM1040 and 27-4 as models for the Roseobacter clade. In the process of locating these genes, we discovered a megaplasmid critical for TDA
biosynthesis that is part of the TM1040 genome, as hereinafter more fully described.

[0034] Materials and Methods [0035] Bacteria and media [0036] The strains used in our study are listed in (Table 1). Silicibacter sp.
TM1040, Phaeobacter 27-4 and Vibrio anguillarum 90-11-287 were grown and maintained in 2216 marine broth or 2216 agar as recommended by the manufacturer (BD Biosciences, Franklin Lakes, NJ).
A marine basal minimal medium (MBM; per liter: 8.47g Tris HC1, 0.37 g of NH4C1, 0.0022 g of K2HPO4, 11.6 g NaC1, 6 g MgSO4, 0.75 g KC1, 1.47 g CaC12=2H20, 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 [0038] Bacterial spent medium was either injected directly (up to 10 L) or purified by mixed phase anion-exchange reversed phase mini column chromatography on Oasis MAX
columns as previously described. Tropodithietic acid was analyzed by reverse phase liquid chromatography (LC) on an Agilent 1100 HPLC system equipped with a diode array detector (DAD). Separation was conducted using a Phenomenex (Torrance, CA) Curosil PFP 15 cm, 2 mm, 3 m column using a water-acetonitrile (ACN) gradient system. Both solvents contained 200 L/L
trifluoroacetic acid, and started 35% ACN increasing this linear to 60% in 6 min. The wavelength 304 4 nm was used for detection. LC-DAD with online high resolution mass spectrometry (HR-MS) using positive and negative electrospray was used for validation of tropodithietic acid detection as previous described.

[0039] Transposon mutagenesis and Tda screening [0040] Electrocompetent roseobacter strains were prepared following the method described by Garg et al. as modified by Miller and Belas. Random transposon insertion libraries were constructed in TM1040 and 27-4 using the EZ-Tn5<R6Kyori/KAN-2>Tnp TransposomeTM Kit (Epicentre, Madison, WI). Strains were spread onto 2216 plates containing kanamycin and incubated for 1 day at 30 C. Individual Kanr transposon insertion strains were transferred to 7x7- arrays on 2216 marine agar plus kanamycin to facilitate further screening. To screen for loss-of-function, antibiotic-negative (Tda ) mutants, a modification of the method described by Bruhn et al. was used. Bacteria were replicated, as a 7x7 array, to a lawn of Vibrio anguillarum strain 90-11-287, and incubated at 20 C for 24 h, after which a zone of clearing indicative of antibiotic production was measured and compared to the parental strain (TM1040 or 27-4). For purposes of this study, 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 30 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 [0042] Bacterial utilization of sole carbon sources was determined by measuring growth in MBM broth that was modified by replacing glycerol with the carbon source to be tested. Carbon compounds tested included amino acids (alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine); sugars (arabinose, fructose, galactose glucose, lactose, maltose, mannose, N-acetylglucosamine, ribose, sucrose, xylose); tricarboxylic acid cycle (TCA) intermediates (citrate, fumurate, succinate); as well as phenylacetic acid and sodium phenylpyruvate.

[0043] Sulfur utilization was tested by growth in MBM containing different sulfur sources:
DMSP, cysteine, methionine, sodium sulfate, and sodium sulfite.

[0044] Bioinformatics analysis [0045] Approximately 1 g of genomic DNA isolated from the candidate mutant was digested with Nco I, self-religated with T4 DNA ligase, and electroporated into DH5a Qpir). Following selection for kanamycin resistance, Kanr colonies were picked and the plasmid isolated for bidirectional sequencing with transposon-specific primers as recommended by the supplier (Epicentre, Madison, WI). Nucleotide sequence thus obtained was analyzed by BLAST analyses using DNA-DNA homology searches again the Silicibacter sp. TM1040 genome (Accession numbers: NC_008044 , NC_008043 , NC_008042). The genes identified are listed in Table 2 for TM1040 and Table 3 for 27-4.
Table 2. Silicibacter sp. TM1040 genes and encoded proteins required for the regulation and synthesis of tropodithietic acid.
GenBank Gene Number Assession Gene Number Designation Function Best Hit Ortholog / E
score ...Y.......... ..;,.... ...Y.... ..............
...,...........................................................................
:...............................................:~
e~~rs~..~ .~.....~...~.... ..a...~......... .~~.. o~
..............::::::::.......:::::::::.......:::::::::.......:::::::::.......::
:::::::

----------------------------------------------------------------------- ---------------------------------- ---------------------------------------------------------------------------------------------------------------------------TM1040_3728 CP000376 paaK Phenylacetate oxidoreductase Roseobacter sp.

phenylacetic acid degradation oxidoreductase PaaK / 8e-161 ---------------------------------------- ----------------------------- ---------- ------------------------------------------------------------------- ----------------------------------------------TM1040_3726 CP000376 paal Phenylacetate oxygenase Roseobacter sp.

phenylacetic acid degradation protein Paal / 4e----------------------------------------- -----------------------------;----------------------------------_-------------------------------------------------------------------------- ----------------------------------------------TM1040_3727 CP000376 paaJ Phenylacetate oxygenase Roseobacter sp.

phenylacetic acid degradation protein PaaJ / 2e-- ----:---------- - - - - - - - - - ----------------------------------------- ----------------------------------------------------------------------EF139203 EF139203 tdaD 4-hydroxybenzoyl-CoA Paracoccus.
thioesterase denitrificans conserved hypothetical protein / 2e-45 ---------------------------------------- -----------------------------............ -------------------------------------------------------------------------------------------------- ---------EF139204 EF139204 tdaE Acyl-CoA dehydrogenase Paracoccus denitrificans PD 1222 acyl-CoA
dehydrogenase /
9e-120 _____ ---------------------------------- ------- - - - - ---_____________________________ EF139201 EF139201 tdaB (3 etherase, glutathione S Paracoccus transferase denitrificans PD 1222 putative (3-etherase ((3-aryl ether cleaving enzyme) protein /
6e-56 --------------------------------------------------=
EF130202 EF130202 tdaC Prephenate dehydratase Paracoccus denitrificans hypothetical .::::~ protein: ~-.2e.: 45-.::::::
11~~~1:1':::~~~~~
~::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>:::
:>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::
::>::::>::::>::::>::::>::::>::::>::::>::::>::::>
:::::::::::::::::::::::::::::::.:.:?
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

----------------------------------------------------------------------- ---------------------------------- ---------------------------------------------------------------------------------------------------------------------------TM1040_2581 CP000377 malY (3-C-S lyase (cystathionase); Roseobacter sp.
amino transferase MED193 aminotransferase, classes I and II /
0.0 ---------------------------------------- ------------------------------------------------------------------------TM10400961 CP000377 tdaH Sulfite oxidase domain Sulfitobacter sp.
protein NAS-14.1 hypothetical protein / 7e 34 ---------------------------------------- ----------------------------- ----------- ----------------------------------------------------------------------------------------------- ----------TM1040_1758 CP000377 cysl Sulfite reductase Roseobacter sp.
MED 193 sulfite reductase / 0.0 ~::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>
::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::>::::
>::::> ::::: ::::: ::::: ::::: ::::: ::::: ::::: ::::: ::::: ::::: ::::: :::::
::::: ::::: ::::: ::::: ::::: ::::: ::::: ::::: ::::: ::::: ::::: ::>:
:~~::::::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:
::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>:::>::::::>::::::>::::::>::::::>
::::::>::::::>::::::>::::
~ :::::::::
;::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
:::::::::::::::::::::::::::::::
~~ EF139205 EF139205 tdaF Phosphopantothenoylcysteine Paracoccus decarboxylase denitrificans flavoprotein / 2e-:
>......... ......... ;
>;
:
.........; .........
........ ....... ........ ......... ........ ........ ........ ......... .
.:., EF139200 EF139200 tdaA LysR substrate binding Paracoccus domain protein denitrificans regulatory protein, LysR:LysR, substrate-binding / le-29 .................................................. ___________________ ________ _______________________________________________________________________________ ____________________________________________ Table 3. Sole carbon source tested for TM1040 and mutants.

Gene Cy Trp Phe Phenylaceti Sodium Sodium 2216 Other s c acid phenylpyruvat phenylbutyra Amino e te acid WT + + + + + + + +
paai + - - - - - + +
paaJ + - - - - - + +
paaK + - - - - - + +
tdaA + + + + + + + +
tdaB + + + + + + + +
tdaC + + + + + + + +
tdaD + + + + + + + +
tdaE + + + + + + + +
tdaF + + + + + + + +
cysl + - - - - - + -malY + + + + + + + +
tdaH + + + + + + + +

[0046] Signature amino acid domains in the deduced amino acid sequence of the respective ORFs were identified using BLASTP, Pfam, SMART, and the Conserved Domains Database (CDD; http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Homologs in roseobacters were identified using BLASTP analysis of Roseobase (http://www.roseobase.org/) and Gordon and Betty Moore Foundation Marine Microbial Genome databases (https://research.venterinstitute.org/moore/) with respective predicted protein sequence as the query sequence and a maximum E value of 1E-30. Homologs in the Global Ocean Sampling Expedition metagenomic libraries (http://camera.calit2.net/index) were identified by BLASTP
analysis using a cutoff E value 1E-20.

[0047] DNA extraction and separation [0048] 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.

[0049] Pulsed Field Gel Electrophoresis (PFGE) 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 C, and a run time of 26 h. Gels were stained with ethidium bromide (EB) and visualized with a Typhoon 9410 (Amersham Biosciences, Piscataway, N.J.).

[0050] PCR amplification [0051] Multiplex PCR amplification was used to screen for the presence of tda genes in Tda mutants. A 716-bp sequence internal to tdaE was amplified using primers 5'-CAGATGATGGTGCCAAAGGACTAT-3'and 5' -GGTCAGTTTCTTCTGCACATACTGG-3' , while (in the same reaction), an interna1401-bp fragment of flaA (accession number: CP000377, locus tag: TM1040_2952) was also amplified using primers 5'-TTGCAGTATCCAATGGTCGTG-3' and 5'-TGAATTGCGTCAGAGTTTGCC-3' as a control.
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 lx reaction buffer (New England BioLabs) with an initial denaturing step at 94 C for 3 min, followed by 30 cycles of 94 C for 1 min each, annealing at 55 C for 30 s, and an elongation at 72 C for 1 min.

[0052] To detect the tdaA-E locus, PCR amplification was conducted with a forward primer complementary to tdaA (5'-CGCTTTCCGGAACTGGAGAT-3') and a reverse primer complementary to tdaE (5' -GGCTGCCGTATAGTTTCAGCA-3' ) 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.

[0055] Following extraction, 100 ng of total genomic DNA purified from each strain was spotted onto a positively charged nylon membrane (Roche). The DNA was cross-linked to the membrane with ultraviolet light using a Stratalinker UV Crosslinker (Stratagene, La Jolla, CA), followed by prehybridization of the membrane at 25 C for 30 min, using the DIG
High Prime DNA Labeling and Detection Starter Kit II (Roche) as described by the manufacturer. The membrane was incubated at 25 C overnight with a double-stranded DNA probe prepared by Hind III digestion of a plasmid bearing tdaA cloned from strain HG1310 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 x15 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.

[0056] RESULTS

[0057] TM1040 produces the sulfur-containing antibiotic tropodithietic acid [0058] 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.
During the course of this investigation, non-pigmented colonies were sometimes seen after TM1040 was incubated on nutrient agar, and subsequent analysis revealed that these `white spontaneous mutants' also had lost antibacterial activity as well.

[0059] TM1040 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. Cell-free supernatants were collected independently from both TM1040 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. 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. Mass spectroscopy of the TM1040 `TDA
peak' was used to confirm the efficacy of the compound as TDA. Taken together, the data corroborate TDA as the antibacterial metabolite produced by TM1040.

[0060] Identification of genes involved in the synthesis of TDA

[0061] With the exception of genes involved in shikimate and phenylacetate metabolism, an analysis of the genome of TM1040 does not provide much insight into genes likely to participate in the biosynthesis and regulation of TDA. To determine the genes required for TDA synthesis, a genome-wide random-insertion transposon bank of 11,284 Kanr colonies was generated in TM1040 and screened for antibiotic loss-of-function mutants (Tda phenotype).
Approximately 0.7% of the transposon insertions (81 out of 11,284) were Tda mutants, all of which were defective in TDA synthesis as well as in pigment formation.

[0062] 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 TM1040 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). It is clear that 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. A thorough analysis of these `orphan' genes is presented below (TDA biosynthesis genes resided on a 130 kb cryptic plasmid).

[0063] 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. Nearly 90% (43 out of 49) did not harbor tdaA-F, as determined by PCR amplification with primers to tdaE, and had lost this DNA
presumably resulting in their Tda phenotype. The transposon insertion in these strains may contribute to the Tda phenotype.

[0064] The sequences obtained from the remaining 6 Tda mutants were highly informative (Table 2).

[0065] An analysis of the genes identified from the 6`genomic' TDA- mutants revealed that the phenylacetate catabolism (paa) pathway is required for TDA synthesis (Fig.
3A). Transposon insertions were identified in homologs of paa7, paaJ, and paaK. The deduced amino acid sequence from each of these ORFs had strong homology to similar proteins encoded by other roseobacters. For example, TM1040 paal is 79% similar to paal of Silicibacter pomeroyi DSS-3, TM1040 paaT is 74% similar to an ORF of Roseobacter sp. MED193, and paaK is 77% to Roseobacter sp. MED193 paaK (Table 2). In other bacteria, 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. The finding that mutations in paa genes affects TDA
synthesis is consistent with the biochemical evidence of phenylacetate metabolism in thiotropocin synthesis.

[0066] 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).

[0067] 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, S032 , S042 , 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).

[0068] TDA biosynthesis genes resided on a 130 kb crXptic plasmid [0069] As previously described, tdaA-F genes were not part of the annotated TM1040 genome and were absent in spontaneous Tda mutants. We conducted a series of bioinformatic analyses to elucidate the potential function of these genes (Table 2) and their proteins. Interestingly, 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. 3B, the orientation and spacing between tdaA and tdaB suggests that these genes form a bicistronic message while tdaC-E are likely to compose an operon separate from tdaAB. tdaF is in a different operon (Fig. 3).

[0070] Amino acid domain identification was useful in assigning potential functions to the encoded proteins. For example, 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.
Table 1. Bacterial strains and plasmids used.

Strain/plasmid Genotype/phenotype Source of reference Escherichia coli F- endAl hsdR17 (rK mK ) supE44 thi-1 (1) DH5a recAl gyrA96 relAl 080dlacZ4M15 DH5a(a,pir) DH5cc transduced with Apir (2, 3) EC100D pir+ F- mcrA D(mrr-hsdRMS-mcrBC) EpicentreTm f80dlacZDM15 DlacX74 recAl endAl araD139 D(ara, leu)7697 galU galK 1- rpsL
nupG pir+(DHFR).

Roseobacters Silicibacter sp. Wild type, antibacterial activity (4) Mutants derived from TM1040 Silicibacter sp. None pigment and tda spontaneous strain current study 1 Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual., 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
2 Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580.
3 Kolter, R., M. Inuzuka, and D. R. Helinski. 1978. Transcomplementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15:1199-1208.
' Miller, T. R., and R. Belas. 2004. Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp. Appl. Environ. Microbiol. 70:3383-3391 HG1005 paaK::EZ-Tn5,Kan "
HG1015 tdaB::EZ-Tn5,Kan "
HG1050 tdaF::EZ-Tn5,Kan "
HG1056 paaJ::EZ-Tn5,Kan "
HG1080 tdaC::EZ-Tn5,Kan "
HG1110 tdaD::EZ-Tn5,Kan "
HG1213 malY::EZ-Tn5,Kan "
HG1220 cysl::EZ-Tn5,Kan "
HG1244 tdaH::EZ-Tn5,Kan "
HG1265 tdaE::EZ-Tn5,Kan "
HG1299 paal::EZ-Tn5,Kan "
HG1310 tdaA::EZ-Tn5,Kan "
Phaeobacter sp. Wild type, antibacterial activity (5, 6) Mutants derived form 27-4 JBB1001 tdaB::EZ-Tn5,Kan current study JBB1003 tdbC::EZ-Tn5,Kan "
JBB1005 tral::EZ-Tn5,Kan "
JBB1006 clpX::EZ-Tn5,Kan "
JBB1007 tdbF::EZ-Tn5,Kan "
JBB1009 tdbA::EZ-Tn5,Kan "
JBB1011 tdbD::EZ-Tn5,Kan "
JBB1029 tdbE::EZ-Tn5,Kan "
JBB1030 tdaA::EZ-Tn5,Kan "
JBB 1044 metF::EZ-Tn5,Kan "

s Bruhn, J. B., L. Gram, and R. Belas. 2007. Production of antibacterial compounds and biofilm formation by Roseobacter species are influenced by culture conditions. Appl. Environ.
Microbiol. 73:442-450.
6 Hjelm, M., O. Bergh, A. Riaza, J. Nielsen, J. Melchiorsen, S. Jensen, H.
Duncan, P. Ahrens, H. Birkbech, and L. Gram. 2004. Selection and identification of autochthonous potential probiotic bacteria from turbot larvae (Scophthalmus maximus) rearing units. Syst. Appl. Microbiol. 27:360-371.

JBB1045 tdbB::EZ-Tn5,Kan Other Roseobacters Roseobacter Wild type, none antibacterial activity (5, 7) algicola 51442 Roseobacter Wild type, none antibacterial activity (5, 8) denitrificans Roseobacter Wild type, none antibacterial activity (5, 8) litoralis 49566 Roseobacter sp. Wild type, antibacterial activity (5, 4) Roseobacter sp. Wild type, antibacterial activity (5, 4) Roseovarius sp. Wild type, antibacterial activity ISM

Roseovarius sp. Wild type, antibacterial activity (5, 4) Roseovarius sp. Wild type, antibacterial activity (5, 4) Silicibacter Wild type, antibacterial activity (5, 9) pomeroyi DSS-Sulfitobacter Wild type, none antibacterial activity (5) 7 Lafay, B., R. Ruimy, C. Rausch de Traubenberg, V. Breittmayer, M. J.
Gauthier, and R. Christen. 1995.
Roseobacter algicola sp. nov., a new marine bacterium isolated from the phycosphere of the toxin-producing dinoflagellate Prorocentrum lima. Int. J. Syst. Bacteriol. 45:290-296.
8 Shiba, T. 1991. Roseobacter litoralis gen. nov., sp. nov., and Roseobacter denitrificans sp. nov., aerobic pink-pigmented bacteria which contain bacteriochlorophyll a. Syst. Appl. Microbiol.
14:140-145.
9 Gonzalez, J. M., J. S. Covert, W. B. Whitman, J. R. Henriksen, F. Mayer, B.
Scharf, R. Schmitt, A. Buchan, J. A. Fuhrman, R. P. Kiene, and M. A. Moran. 2003. Silicibacterpomeroyi sp.
nov. and Roseovarius nubinhibens sp. nov., dimethylsulfoniopropionate-demethylating bacteria from marine environments. Int. J. Syst.
Evol. Microbiol. 53:1261-1269.

sp.1921 Sulfitobacter sp. Wild type, antibacterial activity (5, 10) Sulfitobacter sp. Wild type, none antibacterial activity (5, 11) Vibrio Wild type, serotype 01, susceptible to (5, 12) anguillarum 90- tropodithietic acid Plasmid pSTM3 Harboring tda genes current study pSTM3-1265 pSTM3 carrying a Tn5 insertion in tdaE, current study derived from HG1265 [0071] The involvement of CoA metabolism, addition, or modification is evident from the functional domains on TdaD and TdaE. 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), and TdaF has homology to aldehyde dehydrogenase.

[0072] The secondary evidence suggests that tdaA-F resides on a cryptic plasmid that may be spontaneously lost. To develop a means to test the hypothesis, we used three strains, TM1040, a spontaneous Tda nonpigmented strain of TM1040 (TM1040SM), and HG1265 (tdaE::Tn) (Fig.
5A and Table 1), along with a PCR amplification using primers for tdaA-E, predicted to generate a 3.8 kb product from wild-type DNA. As shown in Fig. 5B, PCR amplification of wild-type 10 Buchan, A., L. S. Collier, E. L. Neidle, and M. A. Moran. 2000. Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine roseobacter lineage. Appl. Environ.
Microbiol. 66:4662-4672.
11 Buchan, A., E. L. Neidle, and M. A. Moran. 2001. Diversity of the ring-cleaving dioxygenase gene pcaH in a salt marsh bacterial community. Appl. Environ. Microbiol. 67:5801-5809.
12 Skov, M. N., K. Pedersen, and J. L. Larsen. 1995. Comparison of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling for typing of Vibrio anguillarum serovar 01. Appl.
Environ. Microbiol. 61:1540-1545.

DNA gave the predicted 3.8 kb band, a 5.7 kb product when tdaE:Tn DNA was used as a template, and no product when the DNA from the SM strain was amplified indicating that the SM strain had lost the tdaA-E locus.

[0073] Total DNA from TM1040, TM1040SM, and HG1265 (tdaE:Tn) was separated by PFGE.
As observed in Fig. 5C, all three strains had high molecular weight DNA, presumably a mixture of chromosomal and pSTM1 and a band or bands at ca. 130 kb, corresponding to the size of pSTM2 (132 kb) (41). Close inspection of this region and comparison between the SM DNA
lane (middle, Fig. 5C) and either the TM1040 or tdaE:Tn DNA (left and right lanes, respectively) shows that the SM band is thinner than either TM1040 or tdaE:Tn hinting that SM
DNA is missing a DNA species in this size range that overlaps with pSTM2.
Repeated attempts to change PFGE conditions did not resolve this region. To overcome this limitation, a Southern blot (Fig. 5D) using a tdaD DNA probe was performed on the gel shown in Fig.
5C, and the results confirmed that the SM DNA, while possessing a 130 kb band, fails to hybridize to tdaD.
In contrast, both wild-type DNA and tdaE:Tn DNA hybridize to the expected band (ca. 130 kb).
This confirms the loss of tda DNA in SM and adds evidence supporting the hypothesis that the missing tda DNA is on a plasmid. It does not rule out the unlikely possibility that tda genes reside on pSTM2 and are somehow deleted from that known molecule.

[0074] To resolve the issue, 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. Consistent with this hypothesis, Southern blotting showed that a tdaD
probe hybridized to a 4.5 kb fragment in wild-type plasmid DNA and to a 6.4 kb fragment from plasmids isolated from the tdaE:Tn strain (Fig. 5E).

[0075] We reasoned that it is possible to transform a cryptic tda plasmid bearing a selectable marker into a suitable host and thereby provide proof of the existence of this plasmid. The transposon that we used, EZ:Tn, contains a kanamycin-resistance gene as well as the oriR6K
origin of replication permitting replication in permissive hosts carrying the pir gene. Thus, the plasmid from tdaE:Tn was used to transform E. coli EC100D (Table 1) with a subsequent selection for kanamycin resistance (Materials and Methods). This transformation was successful despite a very low transformation efficiency resulting in 7 Colony Forming Units (CFUs) per g of mixed plasmid DNA, and provides strong evidence for the existence of a cryptic ca. 130 kb plasmid harboring tda genes. This new plasmid was called pSTM3.

[0076] Twelve random colonies were chosen from the transformation with pSTM3 and the Ncol-digestion pattern of each compared. Fig. 6 shows the four common patterns resulting from this analysis. Although 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.

[0077] Distribution of tda genes in other Roseobacters [0078] The Roseobacter clade produce an antibacterial activity. In light of the current findings, we sought confirmation that other roseobacters had tda genes as well, and chose Phaeobacter 27-4 as a suitable candidate.

[0079] We used the same transposon to construct a 6,321-member library and screened these mutants for the Tda phenotype. 37 Tda mutants were found of which 12 were analyzed further.
Two of the 12 ORFs mutated were similar to TdaA (identity 38%) and TdaB
(identity 55%) from TM1040 (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.

[0080] We also used DNA:DNA hybridization to measure hybridization of a tdaA-F
gene probe to DNA from 14 Roseobacter clade species (Fig. 7). The tda probe hybridized to 8 of the 9 roseobacters that have been established as producing antibacterial activity (Fig. 7), with the ninth, Silicibacter pomeroyi DSS-3, showing a low amount of hybridization.
Three of 6 non-antibiotic-producing roseobacters also positively hybridized to the tda DNA.
This false positive may have resulted from a strain that has very low tda expression and antibiotic activity below the detection limits of the well diffusion assay, or from spurious hybridization to non-tda DNA. The tda probe did not hybridize with DNA from V. anguillarum, implying that the second possibility is the more likely scenario.
Table 4. Phaeobacter 27-4 genes and encoded proteins required for the regulation and synthesis of tropodithietic acid.
---------------------------- ----------------------------------------------------------------- ------------------------------------------------------------ ---------------------------------------------------------------------------GenBank Mutant Assession Gene Number Number Designation Function Best Hit Ortholog / E score ~:~:<i~i.i..i..i..i..i..i..i..i..i..i..i..i..i`..i..i~~i~~i~~i~~i~~i~~i..i..i..
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:~ ~re.e~irs~.rs'':c~a~ d~~~~ :a~d:~
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~ ::::::::::::::::::::::::::: N....
JBB1001 EF139212 (3 etherase, glutathione Sinorhizobium meliloti / tdaB S transferase putative (3-etherase ((3-aryl JBB 1030 ether cleaving enzyme / 4e-52 . ~ 0' r 1~~~~a.1~~~~[;~ :Ai~di~~ o~[:
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... ;...
JBB1044 EF139218 metF 5- Silicibacter sp. TM1040 methyltetrahydrofolate- MetF protein / 2e-77 -homocysteine S-methltransferase -------------------------- ----------------------------------------------------------------- _________________ *
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~ ::::::;::::::::::: <;: ::::::: , JBB1009 EF139215 tdbA D-(3-hydroxybutyrate Roseovarius sp. 217 D-(3-dehydrogenase hydroxybutyrate dehydrogenase / 2e-32 -------------------------------------------------------- ---------------------------------- ------------------------------------------------------------- ----------------------------------------------------------------------------JBB1045 EF139216 tdbB Phosphate Roseobacter sp. MED193 acetyltransferase phosphate acetyltransferase 8e-81 --------------------------------------------------------- ------------------------------------------------------------------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------ ---------------------------------------------------------------------------->
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JBB1003 EF139213 tdbC Lytic transglycosylase, Roseobacter sp. MED193 __________________hypothetical protein / 6e__ 85_____ -------------------------- ----------------------------------------------------------------- ____________ peptidase C14 JBB1005 EF139221 tral Tral, Type IV (Vir- Rhodobacter sphaeroides like) secretion 2.4.1 Tral / 5e-58 ------------- ------------- ------------------------------JBB1011 EF139222 tdbD Type I secretion target Roseobacter sp. MED193 repeat protein type I secretion target repeat protein / 8e-54 --------------------------- ------------------------------ ----------------------------------- ---------------------------------- ------ --------------------------------------------------------------------------------------------JBB1029 EF139216 tdbE Oligopeptide/dipeptide Silicibacter sp. TM1040 ABC transporter binding-protein-dependent transport systems inner membrane component / 6e-------------------------- ----------------------------- ---------------------------------- ----------------------------------------------------------- ------------------------------------------------------------------------------=

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............................................................................, JBB1006 EF139220 clpX ATP-dependent Clp Silicibacter sp. TM1040 protease ATP-binding subunit C1pX
le-47 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - -=
JBB1007 EF139214 tdbF Ribonuclease D Roseobacter sp. MED193 ribonuclease D / 6e-49 -------------------------- -----------------------------= ---------------------------------- ------------------------------------------------------------ ---------------------------------------------------------------------------JBB 1030 EF139217 tdaA LysR substrate binding Paracoccus denitrificans domain protein PD 1222 regulatory protein, LysR:LysR, substrate-binding / 3e-51 -------------------------- ~---------------------------- ~--------------------------------- ~------------------------------------------------------------~---------------------------------------------------------------------------[0081] Distribution of tda genes in the environment [0082] Marine genome and metagenomic databases were searched for sequences with homology to one of the 12 genes (Table 2) required for TDA synthesis by TM1040. While homologs to the proteins involved in phenylacetate and reductive sulfur metabolism were found within the 14 selected roseobacter genomes in Roseobase (http://www.roseobase.org/) and the Gordon and Betty Moore Foundation Marine Microbial Genome databases (https://research.venterinstitute.org/moore/), close homologs of TdaA-F were absent (at a BLASTP E value cutoff of 1E-30). While the reason for the absence of homologs is not known, it is possible, although unlikely, that all 14 roseobacters do not produce TDA, produce an antibacterial activity that involves another compound, or lost their tda plasmid. The last possibility is most likely to have resulted from laboratory culturing, therefore we searched for Tda homologs in environmental metagenomic libraries (http://camera.calit2.net/) that should contain abundant uncultivated roseobacter DNA.

[0083] The data gathered from searching the CAMERA marine metagenomic GOS
dataset database are shown graphically in Fig. 8, where a circle and its relative size indicates the presence and abundance (respectively) of a given protein. As was observed with the roseobacter genomes, phenylacetate and reductive sulfur metabolism proteins were found at numerous sites, with the greatest abundance of PaaIJK and Cysl at site GSOOa, a Sargasso Sea sample (3132'6"
N, 63 35' 42" W). Positive Tda protein `hits' were also recorded in a hypersaline pond sample (GS033) and a sample obtained from Lake Gatun, Panama Canal (Fig. 8). In no sample did we find hits to all 12 proteins involved in TDA biosynthesis.

[0084] Various members of the Roseobacter clade, whose genomes reveal a great potential for the synthesis of bioactive molecules, produce TDA. Many marine bacteria produce an antibiotic activity, including antibacterial activity from roseobacters, e.g., a compound that produces a probiotic effect on scallop larvae and is antagonistic to y-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.

[0085] There is a direct link between the spontaneous appearance of non-pigmented Tda colonies and the loss of pSTM3 of TM1040. Over 40 of the mutants initially screened as Tda were ultimately found to have lost pSTM3. This suggests that loss of pSTM3 is a relatively frequent event during laboratory cultivation of TM1040. Instability of the Tda+ phenotype is not unique to TM1040. The appearance of spontaneous nonpigmented Tda mutants or variants is characteristic of other roseobacters, including Phaeobacter 27-4 and Roseobacter gallaeciensis sp. T5. One possible explanation for the cause of these spontaneous mutants is a loss of a plasmid carrying one or more critical genes required for TDA synthesis.
Indeed, 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.

[0086] Instability of pSTM3 is also apparent when the plasmid is transformed into a nonroseobacter host, e.g., E. coli. As shown in Fig. 6, at least four unique Ncol-restriction fragment patterns were observed from pSTM3 that had been successfully transformed into a new host. As a cause of this instability, it seems improbable that TDA
biosynthesis is to blame, because the pSTM3 used to transform E. coli does not confer a TDA+ phenotype due to the presence of a transposon in tdaE. It is possible that, despite absence of TdaE, some other protein(s) encoded by other tda genes (tdaABCD or -F) may be detrimental when expressed in E. coli. While there is no evidence to directly link instability of pSTM3 in E. coli with spontaneous loss of the plasmid in TM1040, these phenomena may share a common cause. We have initiated efforts to sequence and annotate pSTM3 and compare it to the pSTM3 species obtained from E. coli. Preliminary evidence indicates that pSTM3 harbors a repC homolog upstream of tdaA. RepC forms a complex along with RepAB and is required for plasmid replication and maintenance. It will be determined if the pSTM3 plasmid species obtained from E. coli transformation have defects in repABC.

[0087] The ability of pSTM3 to replicate in E. coli, albeit with significant alteration in the plasmid, suggests that pSTM3 also may transferred to other marine bacteria, perhaps other roseobacters, or even to higher organisms, e.g., dinoflagellates. 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 TM1040 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. Thus, there is a direct correlation between biofilm formation and TDA
biosynthesis.

[0089] Biosynthesis of TDA has several potentially beneficial effects on the 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. In turn, the antibacterial activity of TDA may enhance the growth of TM1040 cells attached to the zoospore by warding off other biofilm-forming bacteria that compete with TM1040 for space on the surface of and nutrients from P. piscicida. Interestingly, 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.

[0090] One of the unexpected results from our study is the paucity of homologous Tda proteins in either the genomes of other sequenced roseobacters or in the CAMERA
metagenomic library (Fig. 8). There are several reasons why 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. In focusing on just the search for Tda homologs in roseobacter genomes, it is possible that, in culturing these roseobacter species in preparation for isolation and purification of their genomic DNAs, the bacteria lost a pSTM3-like plasmid harboring tda genes.
Equally feasible is the possibility that TDA is but one of many antibiotic compounds produced by roseobacters or that more than one biochemical pathway exists to produce TDA. Both arguments may help explain the lack of Tda protein homologs in roseobacter genomes.

[0091] The lack of Tda protein homologs in the marine metagenomics database presents a much more difficult problem to interpret, especially in the context of PaaIJK and Cysl searches that frequently identified their respective homologs in numerous samples within the database (Fig.
8). While the data do not provide definitive answers to this question, our data show that stability and retention of pSTM3 by TM1040 is greatest when the bacteria are directly associated with the dinoflagellate, i.e., the plasmid may be lost when TM1040 is grown in laboratory culture, yet retained when cultivated as part of the Pfiesteria piscicida mesocosm from which the bacteria were isolated. Close association of TM1040 with P. piscicida provides a selection to maintain the pSTM3; that selective pressure is lost when the bacteria are taken away from their host (as happens under laboratory culture). The CAMERA metagenomic samples analyzed were prepared after filtration to remove 0.8 m particles, which may have removed the portion of the roseobacter population harboring a tda plasmid like pSTM3.

[0092] The two metagenomic samples that showed relatively good Tda homolog hits were from a site in the Sargasso Sea and a hypersaline pond, respectively. DMSP is potentially useful by algae as an osmolyte that protects the cells against changes in salinity. Our results suggest that DMSP is not used as a sole sulfur source in the biosynthesis of TDA, and show that there is a correlation between salinity, DMSP, and the presence of Tda homologs.

[0093] The genetic data from the current study, specifically the identification of paaIJK and tdaC (prephenate dehydratase), indicate that TDA biosynthesis originates from the shikimate pathway and proceeds through phenylacetate (Fig. 9). The results also show that phenylacetate-CoA and CoA metabolism is vital to TDA production and are consistent with TdaD-F
involvement in a ring expansion reaction that converts PAA-CoA to a seven-member tropolone ring (step 8 in Fig. 9). TdaB, a homolog of glutathione S-transferase, is a potential agent in the addition of sulfur to the nascent TDA molecule.

[0094] The compounds shown in Fig. 9 include the following:
Table 5 Compound Produced from IUPAC13 name Reaction:
6 1, 2-dihydro-phenylacetyl-CoA

8 2-hydroxy-7-oxo-cyclohepta-3,5-dienecarboxylic acid 9 2,7-dihydroxy-cyclohepta-1,3,5-trienecarboxylic acid 2,7-dihydroxy-3-oxo-cyclohepta-1,4,6-trienecarboxylic acid 11 2,7-dihydroxy-3-thioxo-cyclohepta-1,4,6-trienecarboxylic acid 13 7-hydroxy-2-mercapto-3-thioxo-cyclohepta-1,4,6-trienecarboxylic acid [0095] Identification of a LysR homolog in TdaA is consistent with the regulation of TDA
biosynthesis involving a cofactor. In other bacteria, 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.

[0096] We therefore disclose the genes and proteins required for TDA synthesis by roseobacters, and the occurrence of tda genes on a previously unknown megaplasmid (pSTM3) of TM1040, as aspects of the present invention. 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.
13 International Union of Pure and Applied Chemistry Chemical synthesis of 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.

[0097] Sequencing of the 130 kb pSTM3 plasmid bearing genes required for tropodithietic acid biosynthesis has been carried out with determination of the following sequences.

pSTM3 partial sequence:
contig tdaA-tdaE
GCCCCCCGGGGGGGGGCCCGGGCCAGGTAAATTCGCCCGGGGTTTTACGGGGGGGT
TTTTTTTCCCGAAAGGATGACGCAAAATTCCACCCAGTTTCCTGGCCCCGGAAATAG
AAGCCCCCCGGTTCGGGGGGTGAACTCGGGGGGAGGGGGCCTTTGCCCATCCCAGA
TGCAGCTTGCGCAGATAGGCCGTCGGTTGACCCCCCAAGAGCCAAGCCGCCTCGCCG
GGAGGTGAACTTGCGCTCCCCTTGGCGCTCGGGGGGAAAGGAGGCTTTCGCGTTGAT
TGTGCAATGTGCGCCCAGCCATTCGAAATGCTCCCGAATAAGCTGGTTGAGATCCTC
ATGCAGCGCTTCTGCTGCTGCCGGAGCCTTGGTCGTTGCATGCCGTCCTGCCCTTCGT
ATCCTCTGTGACGGTTCCACTGTGACGGTGGCGATGGCGCAAGGAGCCGCCTCAGAT
CGGGCGTTTTCTCTTCAGCCTGCCCGCCGTGTTCACGGAAATCGACGTTTTTGTATCT
TTCCGTGACTATTTACCGCCGAGCGGGATTCGTGCAAGGGTTTTCTGCCCAAGTTAT
CCACAGGATGCGCAATTTTTGAGCCCCGCAGACGCGGTGATGGCCTCTGGGGGCGG
AGAAGTTGCCTGTCATACCCGTGACACGAGACTAAAGGCATTCTGCAATAGCCAGC
CGCCCAGTCCGGTCTCTCTGTGACCTTTGGCATCCGGGACGGCGCCGCCAAACCGGC
CCCATGTCAGCGCCGCATTGCGGGAAACGCCAGGGCGCAGAAGACCCGAAGACGGC
CCGCAAACCGCCGGATGCCGCGTGGGACAGGGGCGGGACAGGATGGAGAACCGTG
GTGGCCTCGCCCTTTCTGGCGAGGGATTTTCGCGCGTAACCCGTGTGGCGAAACCCC
GCCGAAGCGCGTAAGTCTCAGAAAAAATGACTAAATTATCGGCTTGATAAAATCTG
TAGACGACATAACCTATAGGAGATTCGTTTGCCAGTGTTTTACCCTTGGTTGTTGAG
GGCACATTAATAAGACCGCGGTTCCGGCCAGCTATTGACCGCCGCCGTTGCAGACCC
CCTGCAATGCGCGCCGTCCAGCGAGAGAGACCGACTTTCCCAAAACCCAACCCAAG

ACCAGATATGGTGCACTGTGCGTCATCAGTGAGTGGGAGCGAGATTAGATTTGGAC
ATTCAACAGCTAAGAGTCTTTGTCACCGTTGCAAAACATGGCAGCATCACCCGTGCT
TCTGACATTCTGTGGCAGCCAGCCCTCGGTGAGCGCGCAGATCAAGAGCCTGGAGA
CGACACTCGGGATCACGCTGTTTGAGCGCACCTCGCGGGGCATGGTGGTCACGCAG
GGGGGCGAGCGCCTTCTGGATGAGGCGACCGCTTGTGGATCGGCACAAACAGTTCA
TGCAGGAGGCCTCGCGACTGAAGGGCAGTGTCTCGGGGCTGTTTGCCATGGGCGCA
GGGCGGCATTCGGGCAACGGCTTTGTCAGCTCTTTCCTGCATTGTCTCGGAACGCTT
TCCGGAACTGGAGATCGAGCTCAAACACCTGGCCTCGGCGCAGGTGATCGAGGGGC
TGCGCGATCAGTCGCTGGACATGGGATTTTTCACCGAAACCGAAAGCGACACCTCG
ATTGACGCTGGTGGAGGTGGCCAGTTTCGGCATCTACCTTGCGGCGCCGCGCGGGAT
GATCCGTTGTTCAGAGACCCCTGACTGGGCGCGTCTTCAGGATCAGATCTGGATTGT
CTCGTCTCATGTGGCGCTGCGGTCGCTGGGCCAATGCCCTCATGGAGCAGCATGACA
TTCGCCCAAGGCGGGTGATCAAGGTTGATGACGAGGCGGTGACGCGGACGCTGGTG
GCAAGCGGAGCCGGGGTCGGGCTGTGCATTCTCGGGTGATGAAGCGCTGACGCCGC
CCGATGACATCGACCTGTTGCACCGGGTGCGCAAGACCGCGCGGATCATGTGCGGCT
ATCTCGAAGCGCGCAGCGATGATCCCTCTATTCGCGCGTAGATAATTGGTTCTGGAT
TTGCTGAAATCTCAACAAAAAGGCGAAACACCCTCCTTGTTGCAATTGGCGTAATCA
CAATTTCATTTGAGAATCCCCAAATAAGGGAATACGTCATTCGAGAGTGTTATTTTG
GAGTTGTCATGATTACGATTTATAGCCTCTGTGGCAAAGACGATATTCATTATTCCC
CGCATGTTTGGAAAGTCATTATGGCCCTGCATCACAAAGGGCTTTCATTTGACGTGG
TGCCGTGGATTTTCGACGATCCGCGACATCGAGGGCGGGGCGTTCAACAGCGTGCC
GGTGCTGCGCGATGGCGACCGGGTGATCGGGGACAGCTTCGAGATCTGCACCTATCT
GGATGCCGCCTACCCGGTGCCCCGGCCTGTTTGCCGGTGCGGGCAGTGAGGCGCAG
GTGCGGTTCCTGGAAAGCTATTGCCTGACGGCGCTGCACCCACCGCTCGCGGTGATC
GCGGTGATGGCGATGCATGACATCATGCATCGGGCGATCAGCCTATTTCCGGGCCAA
ACGCGAAGAGCGTTTTGGCGTGTCCATCGAGGCGCTGGCGGAAACCGCGCCCGCCG
AGCGCGCGCGATTGCAGGAGCGGCTGGCGCCGGTGCGCGCCGTCTTACGCATCACA
CTGGCTTGCGGGCGATGCCCCGGCGATGGCCGATTACGTGGTGTTCAGCGCCTTGCA
GTGGTGCTGGGTCGTGGGGCTGCGCGATCTTCTGTCCCCCGACGATTCGGTGGCGCG
TGGTTCAGCCGTGTCAGGCCCTGTTTGGAGGGGCGGCGCAAAAGCCTGCTGGAGCC
CCGCGCTAAGCCTGAGCTGAATCTGCGCGAACAAACCGGCAAAACCCGGCCCAAAT
TCATCTGATGCGCCCCCGATCGGGGCCGCTTTTTGTTGGTTTTGGGGCATTTACGGCT
GTGTCACCAAAGCCGATAGCTGACCTCAGTTTTTCCGAATTGCGACAAAGCGCGTCA
TTGGATCATATGAGTCCCAAGGTTCGATACGTCCTGAGCGAATTGATTTTTGAAACG
GTTGGAAATGAACAAGTAAATGGTTGCGTATCCGAAATTGAATTTCAGTCAATTGAT
GATGCCATTGGAGGACTCTTGAATGGACGTCGCGCTATGGACGGTCCCAGAACCAA
CGCAGTGAAGACATATCCAAAACCTATGACTGGGGTGCGCCATGTTCATACCCTGGG
ACCGGCTGGCACCAACTGTGAAAAGGCGGCGCTGAAATGGGCGGCGCTCAGTGCCG
CAATGCTGCCTGGTCCTGCATGACTCGATGGAGGAGGCCGCAGAGCAGGTCGCGGC
CTGCGGCTGTTCGGTGCTTCTGAGCGTGGTGGCCTACCCGCAGCTGCATTCGATCAT
CTACGACATATCGCGCATCTGGGCTTCTGGATGTGTTCATCATGAAGACCGACGACA
TGGTGCTGGCCTCGGTGAGCGGCGCCATGCCGACGCTGTGCCAGACCC
ACCCGGCGCCGGAAAAGCTGCTGCCGCCCGAGATGCAGCGGATCTATGCGACGAGC
AATTCCCACGCGGCCTCTGAGGTGGCGGCAGGGCGGGGCGATGGCTGCATCACCAC
GCGTGCCGCCGCCGAAGCACGGGCTTTTGGTGGTGCAGACCTTTGGCCAGGTGCCGA
TGGGGTTCACCATTCACGGCCCGCTCAAGCATGCGGGCTGCGCGGACACCGCCTTTG
ACGTTTCAGCACCAGATCACAACAGGATTTTCCCAATGACCCAACGCGCATTTGAGA
CCCGGATCGAAGTCCGCTACCGCGACACCGACTCGATGGGCCATATCAGCAGCCCG

ATCTACTACGACTACATGCAGTCGGCCTATCTGGAATACAGCCGCGCTGCTGGAGCT
GCCGAAGTCCGAAAAGCTGCCGCATATCATGGTGAAAACCGCCTGCGAGTACATCA
GCCAGGCCTATTACGGCGATACCGTGGTGGTGCTGAGCAAAGTGTCGAAATTCGCG
CAAGAGTTTCGAGATCGACCATGAGATCCGCCTTGGCAGCGCGGACGGCCGGGTGG
TGGCAAAGCTACAGTCGGTGCATGTGATGTTCGATTACGAAAAGCAGAGCACCTAC
CCGGTTCCGGAGATTTCGCAGCCGCGTCGCCGATTTTCAGGACGCCGCCTGAGCGCG
CGCCACGGTCCAGAGAGGGAGAATGCAATGGATTTGAGTTGGAGCACGCAGCAGCA
GTCGATCCGGGCGGAGTTTGCCTCCTCGGAGCCGCACAGACCGCGATGAGCTGCGTC
TTGGACGGCGCGCCTTTGACCAGCAGACCTGGGATCAGCTGGGAGAGGCGGGCCTG
TGGCAGATGATGGTGCCAAAGGACTATGGTGGCACCGGGGCGGACCGGGGCTTGCT
GGTGGGATGTCACCGCCGCCCTTGAGGGGCTGGCCTCGACCATCCGCGCGCCGGGG
CTGTTGCTGTCGGTGATCGCCCAAGCGGGTATGGCCTACGCGCTGGAGCTCTTGGCA
CCCGGCGCAGAAATCCGACTATTTCCGCCGCATCCTGCGCGGCGCGCTGAGCGCCAC
GGCCATCGCGGACCCCGACACCGGCACCGATGTCCGCGCCAGCTCCACTTACCTCAG
CCCGCGCCGAACGAACCTTTGTGCTCAACGGGAAGAAATACAACATCGCCCATGCG
CCGGTGGCGAATTTCACTCTGGTGGTCTGCAAGCTCGAAGGCCATGCCCGCGACGGC
ATCTCCCTGGTTCTGGTGGTCAGGACAGAAGGGCGTCACCATCGGTGCCAAGGATCG
CAAGCTTGGAAACCTAGATTTGCCGACGGGGGCGCTCTCGTTTGAGAATGTGCCGCT
GCACTATGGGCATATTCTGGGGGTGCCGGGCAAGGGCTGCGAACCTTGTGCGGTTTG
TCTCGCTGGGGCGGATCTATTACGGGCTGGTGGCGGCGACCCTGTGCGGCCCGATGC
TTGCGGAGGCGCTGTCTTATGCCAAGGCCCGGCAGACCTTTGGCAGCCCATCGTGAT
CACCAGTATGTGCAGAAGAAACTGACCGATATGCGCATCGCGGCAGAGACCGCCAA
ATGGACCTCTTATGGGGCGTTGCACCAGTTGCTGAGCGGCGCGCCCGAGGCGGTGA
GAGCTGTTCGATGCCAAGCTGGCCGGAGCCAGCGCGATCACCGATGGGGCCGTGGA
CCTGCTGAAACTATACGGCAGCCGGGGCTATCACGTAGGGCGAGGTGTCCACGTTCC
TGCGCGATGCGCTGCCTTTTGCAGCGTGGCGGCACCGAGGAAATGCATCGGCGCAA
CATCATGAACCAGATGATGCGAGAGGCCCGCCCGGCCAAGTCCAAGCCCGCCGCCC
CGGCGCGGGATCTGGAAACCGTCTGAGGCCGCCTTTATTGATTGGAGACAATCATGT
TAAAAGATTTCAACAGCTTGCGCTGTCTCGCGCATGGTGCTGCACTAGGCGCTGTAC
TGGGCGCGATGCCGCTTGCGGCGGGTGCCGCAGAAGAGGGATCCTGAGCGAGGCCA
GATCGACTGGGCCGCCGCAGAGGACTCGGCGGTGGCAACCGCTACAGCAGAAGCCG
CACTCACGGAGGCGTTTCTGGCCCTGCCCGCGAGCGCCGAGCCCACCGGTTTGCGGT
GATGCTCTTTGGGCGGCGGCGGATCTGCCCGAGCCGGGCTTTGTCAGTCAGGGCAGC
GCCTATGTGGCCTATTACGCGCAGGACGATATTCAGCTTTCGATCTCGGGGTCAAAG
GCGGTGGTCAGGCGGGGGATGCGCTGTTCTGCACCATGCCCCAAGCGCGTGGGAAA
GCATCGGAACGGGCGCGGATTACAAT

#is# tda A 4d:"i B tda C t~~ D tdaE
.~
.......... .......... .......... .................
:iiiii< ;

tdaF and membrane protein gene:
GCGCCGGTGGAGAGAACAAACATCCTCGCGGCTTGCTGGAGTTCTACCTCGAGGAA
GCGAGATCACGGCCCGCCACCTGCTGACACAAGTCTCGGTATCTGAGTCGAAGGTGT
GCCGGTGCGCGAGGGAACACCCGTTTCGCAAACCGCTCTCCCGGCTCAGGGTGGGG
GATCACCTGCGCAGCGCCGACCGCATCGTGCGGCCGAGGACGTCGAAGCCTTTGGC
CACCTCACGGGCGATCTGTTTTATGCCCTTGGACGAGGCCGCCGCGCGCAATCATCC
GTTCTTTGACGGGCGCGTCGCGCATGGGCAATACATCATGGCGCTGGCCAACGGCCT
CTTTGTGGACCCCGAGCCCGGCCCCGTGCTGGCCAATCTCGCCCCGCGATCTGCGTT
TTTTTGCGCCGGTCTATTTCGACACCGCGCTCTATGTGACGCTCACCTGCTGCTGCAT
CGGCCCCCTCAACAGGTCGGGCGCGGCCGAAGTGCAATGGAGCTGCCAGGTCGGGC
AGCGATGACGACACCCGCGTGGCCCAGTTCGACCTGCTGACCCTTGTCGCCGCCCAA
TGGCCGCCCCAGCCCGCCCCCCGCGCCTGAGAGGCCCGAGAGGCCTGAGACGCCGG
GTACGCCAATGCCCCTTCCCCTGCTATTGAAACAAAAGGATTTCCAATGACCTCTGC
TCCAAAGCCCCGCATCCTGATCGGTGCCTGCGGCTCGCTCGACCTGCTGATGCTGCC
GCAGCACCTGCGCGCCATCAGGACACATCGACTGCACGCTGAGCCTGATGCTCACGC
CGACGGCGGTGAAATTTGTCAACACGGATGCGCTGGCCCTGCTGGTGGACCGGCTG
ATCCACGGCGACCGCCCCGACGACTGGCCACGCACAAGCCGGACGCCTTGCCGCCG
ATCACGATCTTCTTGCGGTGCTGCCGACAACCGCAAACACCCTCAGTGCCGTGGCCA
ACGGCAGCTCGCAGAACCGCCTCACCACGGTGATCCTCGCAGCGGATTTCCGGACTG
TTCTTTCCCGTGATGGGCGGGCCGATGTGGGACAAGGCCTCGGTGCAGCGCAATGTG
AGCCAGATCCGCGCCGACGGGTATGAGGTGTTTCAGCCGGTCTGGCGCGAACACAG
CGCCCGCATCGCAAAAGGTCCACGGCCATCATTCGCTGCCGGACCCCGCGGATGTGG
TCGACATCCTCCAAAGCCGCCTGCCCGCGCAGCGCTGACCCGGCCTACCCGCCTGCC
CCCCCTGCCCCACAGATCTGTCCAAACAGGAAACGCCGCCGGATCTCCTCCGGCGGC
GTTTCTCGTGGTCTCTTTGCCTTTGGCCCTAGCCGGTCACATCACGCAGGCCGGGGC
GCAGCATGGGCCACAGCCGCGCCTGCCCAGCGCCGCAGATAGAGCCCCACCCCAAA
GGAGAGATGCGTCATCGCGCTCTTCAGCTGCGCAAAGGTCGGATCGGGCTTGTTGCT
GGCCATGATGCCCGCACCCATCGCCGGCTGCATCACAAAAAGGGAAAGACATGGTG
CCGAGCCCCACCACAAGCGCCAGCAACACCTGCGGCCGTTGCAGCTGCCCGACGCC
CCCGATCGCCACAAAGAGCGCCGCAAAGACCACGCCCACCGCATAATGTACCGCCA
GCCAAGCGCGCCTCACCCGCCACCGGCGCCGCCGCGCGAATGCTCTCATGGGCGAA
CACGCCCTCGGGCATATGGCCGACCCAACGCCCCACCAGGGCAAAGTTGCTCTGCG
GAATCGCAAACAGCGCCTCCGCCAGCACGGCCCAGAGATCCATCACCACCGTGGCA
CCCACGCCCAAGCAGTACGCGAAAAACAATCTGTGCAGGAGACATCCTGAGAACCC
TCTCTCTCGCTCACAGGCGCCAGCCGCCTGGCCAGCCCCTTTTGAAGATGCGCCCGA
CCCATCAAAATGCGGGCCGGGCGGTACGCGTTACGCCCTTTACTGGGACACCATCGT

GCTATCGAACCCGGCACAGGTCTGGGGCGCGGCGGCCTCGGCGCTGAGGGGTTGTA
GCGCGGTTGCAGCGACGTGATGCCCGATCTGGTAGCCACATATCGCCCACCGCGTTC
GAGCGCCGGTAATGCACGCCGCCAATCAGGCGCGATTCCGATTCCTCTTCGGCGAGG
TGCTGCAGACTGTCGTAAGACTTGCTGATGTTGGCGGCCCCCTCAAAGGGACGGGA
AAGGCGCGCTGCCAAAGACCGAGGTCATCACCTCAAGCGCTGCGCTGCCGCTGGTG
CAATGCTGACAGGGGTATTCGGGATGCATCGGCGCAGGAATGCGCGATTCCCAGAC
CAGACCGGCTCAGGTCGGGGTGGTTGTAGGTTTTGCCCAACGCCTCCACGGCGGTCT
GTGGCCGCCAGAGCGCGTAGCTGTACTTGGCGTTGAACCCGGCCACCAACGCATCGC
TCAGGGCGACATTCAACACGCCACATGCGCGTTTCCTCGAGAAGAGGCAGCGCGCC
AGGCCGCGCAGAG

F uta3iwe memÃarance Proteita '{Kb trfaF Puiative PhosphOgsterase ~ \ : ._ <<' ~;;:?;??>::?>::?>::?>:::>??>:<<
s~ f i 3:i 1030+cQ ltEg 1377 071119, lipoprotein:

GAATTCGCCCGCCTTGATCGCGGCGCGCACCTCTTCCATCGGGGCGATGGCCTCTTC
GGGCACCATCGACGCGGTAAAGGAGATGTCGTTGCCGCCCTCTTTCATGAGGCCAA
AGCGGGTGTAGTCCCCCGCTGGAATTGCCCGCCGCGCGATCCGCCAGCGCCGCCCCA
AGGATGGGGCCAAAGTTCCAGATCGCATTCGAGAACACGGTCTCGGGGTAGCGCTC
GGTATAATCGGTGAGCGAGCCCACCGCTGATGCCGCGTTCCTTGGCGGCGTCGGCGG
TGCCGATCCGCTCGCCAAAGAGGATGTCGGCGCCGGAATCGATCTGCGCCAGACCG
GCCTCGCGGGCCTTGGGCGGATCAAAGAAGGTGCCGATAAGTGATGAGATGGGTCG
CATCGGGACGCACCGCGTCGACACCCTGACGGAACCCGTTGATGAGCATGTTGACCT
CGGGGATCGGGATCGCCCCCACCGCGCCAAAGACGCCGGACTGGCTCATCTTGCCG
CAGCATGCCGCACAGATAGGCCGCCTCGTGGTTCCAGGTGCCAAAGGTGCCAAAGT
TGTCGCCCGCGGGCTTGCCGCTGGAGCCCATCACGAAGCGCGTGTCGGGATAGTCGC
CCGCCACCTGCGCGCTCGCGCTCCACCGCATAGGCTTCGCCCACGATCACATCCGCG
CCCTGCTCGGCATATTCACGCATCGCGCGCGCATAGTCGGTGCCCGCGATCCCCTCG
GAAAAGACATATTCGATCTCGCGCGCTTGCCGCTTCGAGCATCGCCACATGCAGACG
CGAGTTCCACGCGTTCTCCACCGGCGAGGCATGCACCCCTGCCACCTTGATCGGCGC
TTGCGCCAGCACCATCGGCGCCGGCAGCAACGAGGCCGCCCCAAGTGCTGCGCCGC
TCTTCAGGATCGATCTTCGGGTTCATACC
repC:

GAATTCCTCGGCGCGGACATCCTCTGCCAGCATTTCGATCTCTTGCGCACGCAGCCG
CAGGGGAGAGAGATCAAAGCCATAGGCGACCTTCTCGGTGCCATAGCGCCGCGCAT
AGCGTTTGCCATTGGCTGTCGCGTCTGAGCAGCAAACCGGCCTCGACCAGGCGGGCG
AGATAGCGGCGCATGGTGGAATTGGCCATGCCGTTGAGCCGCTCGCAGATGCTCTG
GTTCGAGGGGTGAATGACAAGGTCGCGTCACGGGCAGCTCGGCCCCGGACCAGAAG
CTCAAGAGCGCTTGCAGCACTGAGAGATCCCGGTCGCTGAGGCCAAAATGATGCCG

GGCGGTCGCGAGATCGCGCAGCACATTCCACTTGCTGACAGTGAAGTGCGGTCGTG
GGCTGAGACCTCGGGGCCTCGGGATCGGTGGATGCGGCAGGCATACGGGAGCTGGC
CGCCTGAAGCTGCGTCTGACGTTTGATCAGAACAGCATCAACGGTGCGCCCAAAAG
CGGACAGGATGATACCCCATGTTTCATTCACGAAGACAAAGAAATCCCGTTCTGCGA
ATCACATTTGACTTGCAGTTTCAGGCTCCTGACACTAGCTTGATGGTGCTAAACACA
AGTCAGGGTCTGTGGGCGATGTCTTTGCGGGACCTTTTCTTTTGTCTGCTCGTGCCTC
CTTTCTTAG

[0098] 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.

[0099] A still further aspect of the invention relates to a method of purification of TDA by HPLC techniques.

[00100] An illustrative purification technique is set out below.
Purification of Compound.

1. Roseobacter 27-4 was grown in 500 ml MB in a 5 liter volumetric flask at 25 C for 4 days.

2. The cells were removed by centrifugation (10,000 x g for 10 min).
3. The pH of the supernatant was adjusted to 3.5 4. Extraction was carried out with 3 times 500 ml ethyl acetate acidified with 0.1 Io formic acid (FA) 5. The organic phase was transferred to a vessel and evaporated to dryness under nitrogen flow.

6. The dry ethyl acetate extract was redissolved in 3 x 3 ml acetonitrile (CAN)-water (1:19) containing 1% FA

7. The redissolved extract was sequentially applied to two 60 mg Oasis MAX
columns (Waters, Milford, MA) which had previously been sequentially conditioned with 4 ml methanol (HPLC grade) and 3 ml CAN-water (1:19) containing 1 Io FA.

8. After loading the samples by gravity the columns were washed with 4 ml PBS
buffer (pH
7).

9. 3.5 ml CAN-water (1:1) was passed through the column and collected (fraction 1) 10. 3.5 ml CAN-water (9:1) (fraction 2) 11. 3.5 ml CAN-water (1:1) with 2% FA (fraction 3) 12. 3.5 ml CAN-water (9:1) with 2% FA (fraction 4) 13. The solvents were then removed in vacuo on a SpeedVac (ThemoSavant, Holbrook, NY).

INDUSTRIAL APPLICABILITY

[00101] 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.

Claims (22)

1. An isolated nucleic acid encoding a megaplasmid (pSTM3) of Silicibacter sp.
TM1040;
wherein the nucleic acid comprises genes involved in tropodithietic acid biosynthesis of Roseobacter bacteria.

2. The isolated nucleic acid of claim 1, having a nucleic acid sequence comprising SEQ. ID. 1, SEQ. ID. 2, SEQ. ID. 3, and SEQ. ID. 4.

3. The isolated nucleic acid of claim 1, having a nucleic acid sequence comprising SEQ. ID. 1.

4. The isolated nucleic acid of claim 1, having a nucleic acid sequence comprising SEQ. ID. 2.

5. The isolated nucleic acid of claim 1, having a nucleic acid sequence comprising SEQ. ID. 3.

6. The isolated nucleic acid of claim 1, having a nucleic acid sequence comprising SEQ. ID. 4.

7. 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.

8. The protein of claim 7, wherein the nucleic acid sequence represented by SEQ. ID. 1 is selected from the group consisting of tdaA, tdaB, tdaC, tdaD, and tdaE.

9. 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.

10. The protein of claim 9, wherein the nucleic acid sequence represented by SEQ. ID. 2 is tdaF.

11. 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.

12. 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.

13. An antibacterial composition comprising tropodithietic acid isolated from bacteria of the Roseobacter clade.

14. The antibacterial composition of claim 13, isolated from Silicibacter sp.
TM1040.

15. The antibacterial composition of claim 13, isolated from Roseobacter sp.
27-4.

16. A method for producing an antibacterial composition comprising tropodithietic acid, the method comprising:

a) culturing Silicibacter sp. TM1040 in a culture medium supporting growth of the bacterium and production of tropodithietic acid; and b) separating the tropodithietic acid from the culture medium; and c) purifying the tropodithietic acid by high performance liquid chromatography.

17. A method for producing an antibacterial composition comprising tropodithietic acid, the method comprising:

a) culturing Roseobacter sp.27-4 in a culture medium supporting growth of the bacterium and production of tropodithietic acid; and b) separating the tropodithietic acid from the culture medium; and c) purifying the tropodithietic acid by high performance liquid chromatography.

18. A method of treating or preventing bacterial disease in an subject in need of such treatment or prevention, comprising administering to said subject an antibacterial composition comprising tropodithietic acid isolated from bacteria of the Roseobacter clade.

19. The method of claim 18, wherein the tropodithietic acid is isolated from Silicibacter sp.
TM1040.

20. The method of claim 18, wherein the tropodithietic acid is isolated from Roseobacter sp. 24-7.

21. The plasmid pSTM3.

22. A compound selected from the group consisting of:
1, 2-dihydro-phenylacetyl-CoA;
2-hydroxy-7-oxo-cyclohepta-3,5-dienecarboxylic acid;
2,7-; dihydroxy-cyclohepta-1,3,5-trienecarboxylic acid;
2,7-dihydroxy-3-oxo-cyclohepta-1,4,6-trienecarboxylic acid;
2,7-dihydroxy-3-thioxo-cyclohepta-1,4,6-trienecarboxylic acid; and 7-hydroxy-2-mercapto-3-thioxo-cyclohepta-1,4,6-trienecarboxylic acid.