Article Figures & Data
- FIG 1
Model for algal-bacterial symbiosis involving P. inhibens and E. huxleyi. The symbiosis comprises two modes, a mutualistic phase (green arrows) and a parasitic phase (red arrows) (2). In the mutualistic phase, phenylacetic acid (PAA) provides a precursor for TDA, and both metabolites serve as beneficial molecules to the algae, which provide the bacteria with food in the form of DMSP. In the parasitic phase, the senescing algal host releases pCA. The bacteria respond by combining fragments of DMSP, PAA, and pCA to synthesize the algaecide roseobacticide A (23).
- FIG 2
Monitoring roseobacticide production by fluorescence spectroscopy. Samples were excited at the absorbance maximum of roseobacticides (430 nm), and emission was monitored between 450 and 700 nm. Shown are emission spectra for YTSS medium with sinapic acid (black trace), cultures of P. inhibens in YTSS in the absence of inducer sinapic acid (blue trace), and cultures of P. inhibens in YTSS in the presence of sinapic acid (red trace). (Inset) Fluorescence emission spectra of wt P. inhibens (red trace) and a P. inhibens Tn mutant (Tn5::PGA1_262p00840) that is deficient in roseobacticide synthesis. Both were grown in the presence of 1 mM sinapic acid in a 96-well assay plate. The emission trace of wt P. inhibens grown in the absence of sinapic acid has been subtracted from both spectra to remove fluorescence emission properties not associated with roseobacticide production (normalized fluorescence [Norm. Fl.] intensity).
- FIG 3
Biosynthetic genes required for roseobacticide production. (A) Transposon insertions into the tda and paa operons and the patB gene, which result in a roseobacticide-deficient phenotype. Black arrowheads indicate the sites of transposon insertion. (B) HPLC-MS results for the extracts of wt P. inhibens and selected mutant strains in the presence of sinapic acid. The starred peak in the wt trace represents roseobacticide B. All targeted knockout mutants failed to produce roseobacticide. The traces have been vertically offset for clarity.
- FIG 4
Involvement of PatB in roseobacticide biosynthesis. (A) Reaction catalyzed by PatB in the presence of cofactor PLP. (B) Proposed mechanism for PatB. See the text for further details. (C) Proposed pathway for incorporation of the DMSP sulfur into roseobacticides and TDA via PatB. Conversion of DMSP into Cys has been described (step a) (38). A cystine reductase may generate cystine from Cys (step b). PatB converts cystine to pyruvate and S-thio-Cys (step c). TdaB and TdaF may be involved in the insertion of S from S-thio-Cys into roseobacticides and TDA (step d) (29).
- FIG 5
Working model for roseobacticide biosynthesis. (A) Two pathways can deliver the troponoid precursor for roseobacticides, the paaN pathway (blue arrows) or the paa catabolon pathway (black arrows). Our results are consistent with the involvement of the paa catabolon, which through the action of PaaABCDE delivers ring-1,2-epoxyphenylacetyl-CoA (compound 8) from phenylacetyl-CoA (compound 7), which is further elaborated into an oxepin-CoA (compound 9) via PaaG (30, 31). A bifunctional PaaZ could generate 3-hydroxy-5-cycloheptene-1-one-2-formyl-CoA (compound 11) via 3-oxo-5,6-dehydrosuberyl-CoA semialdehyde (compound 10), which through possibly spontaneous loss of water would furnish intermediate compound 12 (2,5-cycloheptadiene-1-one-2-formyl-CoA). This may serve as a substrate for thiol insertion putatively via TdaB and TdaF to give the β-thiotropone analog (compound 13) (29), possibly the last common intermediate in the TDA and roseobacticides pathways. Addition of another thiol, oxidation, and hydrolysis of the CoA-thioester would give the reduced form of TDA, while reaction with a glyoxylyl-CoA (compound 14) and further modifications would give roseobacticides. The orders of these latter steps are not known in either pathway. (B) Highlighted is the β-thiotropone substructure within both TDA and roseobacticide B. This shared moiety might explain the common biosynthetic origins for these structurally and functionally distinct molecules.
- FIG 6
Roseobacticide biosynthesis is regulated by quorum sensing via 3-OH–C10-HSL. HPLC-MS profiles are shown for extracts of wt P. inhibens and selected mutant strains in the presence of sinapic acid (SA). The peak in the wt trace corresponds to roseobacticide B. The ΔtdaA and ΔpgaI strains individually failed to produce roseobacticides, while a coculture of these mutant strains, or a ΔpgaI strain culture supplemented with 3-OH–C10-HSL, generated roseobacticides. The traces have been vertically offset for clarity.
- FIG 7
Preliminary regulatory model for roseobacticide production. Single arrows indicate biosynthetic processes or small-molecule secretion, while double arrows connote induction of gene expression. Growth in the mutualistic phase at high cell densities leads to production of 3-OH–C10-HSL via PgaI, a homoserine lactone synthase (step a) (42). At threshold concentrations, this signal positively regulates pgaR/pgaI gene expression (step b) as well as expression of tdaA (step c), which induces the tda cluster and leads to production of TDA (42), a molecule that we propose protects the algal host (steps d and e) (2). However, in the parasitic phase, algal p-coumaric acid, or other phenylpropanoids, such as sinapic acid (2, 11), induce roseobacticide-specific genes (step f), which in conjunction with the tda operon give rise to roseobacticide (steps g and h), a potent toxin that kills the algal host (step i). Note that pCA serves as a precursor and an inducer for some roseobacticides, such as roseobacticide A (see Fig. 1) (23), while for other analogs, such as roseobacticide B or C, it acts only as an inducer.
- TABLE 1
Putative biosynthetic genes required for roseobacticide production by P. inhibensa
Metabolic pathway and locus tag Gene % relative productionb Predicted functionc Phenylacetate catabolism PGA1_c04090 paaJ 0 β-Ketoadipyl-CoA thiolase PGA1_c04080 paaA 0 Ring-1,2-phenylacetyl-CoA epoxidased PGA1_c04060 paaC 0 Ring-1,2-phenylacetyl-CoA epoxidased PGA1_c04040 paaE 0 Ring-1,2-phenylacetyl-CoA epoxidased Sulfur metabolism PGA1_c20760 cysI 4 Nitrite/sulfite reductase PGA1_c00860 patB 0 Cystathionine β-lyase TDA biosynthesise PGA1_262p00980 tdaA 0 LysR transcriptional regulator PGA1_262p00970 tdaB 0 β-Etherase PGA1_262p00960 tdaC 0 Prephenate dehydratase PGA1_262p00950 tdaD 0 Thioesterase PGA1_262P00940 tdaE 0 Acyl-CoA dehydrogenase PGA1_262p00800 paaZ2 0 Enoyl-CoA hydratase PGA1_262p00840 0 PUFf ↵a See Table S1 in the supplemental material for a list of all other genes identified using the Tn mutagenesis screen.
b Production with each Tn mutant, relative to that of wt, determined using an HPLC-MS assay for direct detection of roseobacticide B.
↵c Predicted functions are based on protein homology to proteins in the IMG database.
↵d See reference 31.
↵e See reference 28.
↵f PUF, protein of unknown function.
Supplemental Material
Figure S1
Extinction coefficient of purified roseobacticide B as a function of wavelength. Download Figure S1, EPS file, 1.5 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
P. inhibens genes required for roseobacticide biosynthesis, identified by transposon mutagenesis. Table S1, DOCX file, 0.02 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
NusA-PatB activity assay results using DTNB. Shown are extracted ion chromatograms in negative ion detection mode for m/z 198.0 (the aryl mercaptide product of DTNB). This product was detected in a reaction mixture containing NusA-PatB, cystine, and PLP (red trace), but not when NusA-PatB was omitted from the reaction mixture (blue trace). A reaction mixture lacking cystine but containing the other components also failed to produce the aryl mercaptide product (data not shown). Download Figure S2, EPS file, 1.7 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
NusA-PatB activity assay based on a coupled LDH reaction for detection of pyruvate. Shown is NADH consumption as a function of time. When all reaction components are present (red trace), NADH is consumed, indicative of formation of pyruvate and its conversion to lactate via NADH and LDH. When NusA-PatB is omitted from the reaction mixture (blue trace), NADH is not consumed. Likewise, no NADH is consumed when other components are omitted, e.g., cystine or LDH (data not shown). Download Figure S3, EPS file, 1.3 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Time course of roseobacticide production in the absence and presence of supplemented QS signal (3-OH–C10-HSL). Symbols represent the optical density at 600 nm at various time points, while bars represent the levels of roseobacticide. Black symbols and bars are results for wt P. inhibens cultures in the absence of the QS signal; red symbols and bars represent results for P. inhibens cultures grown in the presence of 1 µM 3-OH–C10-HSL. Relative roseobacticide levels were determined by extracting each culture with ethyl acetate and analyzing the dried organic layer by HPLC-MS, as described in Materials and Methods. The levels of the two most abundant analogs, roseobacticides B and E (identified by retention time and mass spectrometry), were determined by integration of the corresponding peaks at 430 nm and subsequently plotted as a function of time. Download Figure S4, EPS file, 1.2 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Primers used to generate and validate targeted P. inhibens knockout mutants. Table S2, DOCX file, 0.02 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
- Figure sf1, EPS - Figure sf1, EPS
- Figure sf2, EPS - Figure sf2, EPS
- Figure sf3, EPS - Figure sf3, EPS
- Figure sf4, EPS - Figure sf4, EPS
- Table st1, DOCX - Table st1, DOCX
- Table st2, DOCX - Table st2, DOCX
