FIGURE LEGENDS
Fig. 1. Genetic map and putative pathway responsible for carotenoid biosynthesis by P. ananatis PA13 (CP003086) andP . agglomerans Eho10 (M87280). (A) The carotenoid gene cluster of P . ananatis consisted ofcrtEXYIB and Z ; and that for P . agglomerans ofcrtEidicrtXYIB andZ . Gene numbers were shown on the carotenoid gene map. (B) The putative carotenoid biosynthetic pathway of P . ananatisinferred according to the pathway of Pantoea species (Misawaet al ., 1995) and plants (Guerinot, 2000). The involved enzymes include isopenthyl diphosphate (IPP) isomerase encoded by idi,geranylgeranyl diphosphate (GGPP) synthetase by crtE , phytoene synthase by crtB , phytoene desaturase by crtI , lycopene β-cyclase by crtY, β-carotene hydroxylase by crtZ , and zeaxanthin glucosyl transferase by crtX .
Fig. 2. Confirmation of transcriptional units in the carotenoid gene cluster of P . ananatis by RT-PCR. RT-PCR products were confirmed by Southern hybridisation. Black arrows indicate the extension and transcription directions of the crtEXYIB operon andcrtZ gene. An arrow below the open arrows represents the product of RT reactions. The short thick bars below the RT arrow indicate the PCR products from the corresponding RT reactions. The expected sizes of the PCR products are indicated below the labels. Agarose gel analysis (upper panel) and Southern analysis (lower panel) of the RT-PCR products of the crtEXYIB operon and crtZ gene. Southern hybridisation was performed using pCOK128 as a probe. Lanes 1–3, 4–6, 7–9, 10–12, and 13–15 correspond to the products of PCR1, PCR2, PCR3, PCR4, and PCR5, respectively. Lanes 1, 4, 7, 10, and 13: PCR products from the DNA template as positive controls; lanes 2, 5, 8, 11, and 14: PCR products from the RNA template as negative controls; and lanes 3, 6, 9, 12, and 15: RT-PCR products.
Fig. 3. Production of phytoene, lycopene, and β-carotene inE. coli. HPLC analysis of phytoene (A), lycopene (B), and β-carotene (C) production. a, E. coliDH5α/pYS71(pBBR1MCS5::crtEB) producing phytoene (retention time 2 min, 280 nm); b, E. coliDH5α/pYS69(pBBR1MCS5::crtEBI) producing lycopene (retention time 11 min, 470 nm); and c, E. coliDH5α/pYS76(pBBR1MCS5::crtEBIY) producing β-carotene (retention time 14.8 min, 450 nm). HPLC analysis confirmed that the E. coli strains harbouring pYS71, pYS69, and pYS76 produced phytoene, lycopene, and β-carotene, respectively. (D), Colour change of harvested E. coli cells harbouring pYS71, pYS69, or pYS76. The harvested cells showed colourless phytoene, magenta lycopene, or orange β-carotene. PS, LS, and CS indicate the phytoene, lycopene, and β-carotene standards, respectively.Fig. 4. Carotenoids confer toxoflavin tolerance to P.ananatis. (A) Construction of the crtE::pCOK184 mutant and complementation plasmid pCOK218. − or + indicates negative or positive carotenoid production, respectively. (B) Toxoflavin tolerance ofP. ananatis. The wild-type and crtE::pCOK184 mutant carrying pCOK218 exhibited greater toxoflavin tolerance than thecrtE mutant; however, the crtE::pCOK184 mutant was more sensitive than the wild-type to toxoflavin at 20 µg mL−1. Pantoea ananatis PA13 is sensitive to toxoflavin concentrations > 20 µg mL−1.
Fig. 5. HfqArcZ → RpoS Ͱ ClpXP regulatory networks. (A) An illustration showing the HfqArcZ → RpoS Ͱ ClpXP regulatory networks based on E . coli (Rajuet al ., 2012). RpoS is regulated positively by Hfq and its cognate sRNA ArcZ, and negatively by ClpXP. RpoS-dependent carotenoid production in P . agglomerans (formerly Erwinia herbicola ) was reported previously (Becker-Hapaka et al ., 1997). (B) Carotenoid production in the wild-type (W), ∆rpoS,hfq,arcZ,clpP , and complementation (+) strains. Values are means ± standard deviation (SD) of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. wild-type.
Fig. 6. EanR negatively regulates carotenoid productionvia inhibition of rpoS . (A) QS signal production of the wild-type and ∆eanI,eanR , and ∆eanI‒R mutants as well as the ∆eanI‒R mutant carrying pCOK199 on C .violaceum CV026 biosensor-embedded plates. (B) Carotenoid production of the wild-type and ∆eanI,eanR , and ∆eanI‒R mutants as well as the ∆eanI‒R mutant carrying pCOK199. (C) Quantification of carotenoid production of the PA13 derivatives. Carotenoid production was identical to that shown in (B). Values are means ± standard deviation (SD) of three independent experiments. ***p < 0.001 vs. wild-type. (D) β-Galactosidase activity reporting rpoS expression. rpoS expression was induced in the absence of EanR and decreased in the absence of EanI, indicating that EanR negatively regulates rpoS expression and QS signals de-repress EanR. Values are means ± standard deviation (SD) of three independent experiments. ***p < 0.001 vs. PA13L. (E) Genetic map of rpoS locus and putative lux box. Inverted repeat sequences are shown in bold.
Fig. 7. Hfq regulates the expression of eanI QS signal synthase. (A) Characterisation and quantification of AHL signals in wild-type (W), ∆hfq mutant (−), and complementation (+; pCOK335) strains of P . ananatis PA13. The culture supernatants of the PA13 derivatives were extracted with ethyl acetate at OD600 values of 0.9, 1.5 and 1.8. Ethyl acetate extracts were applied to C18 reversed-phase thin layer chromatography (TLC) plates. AHL signals were visualised with theC . violaceum CV026 biosensor, and synthetic C6-HSL and 3-oxo-C6-HSL were used as AHL standards. (B) 3-oxo-C6AHL signal production of the wild-type (W), ∆hfq mutant (‒), and complementation strain carrying pCOK335 (+; pLAFR3::hfq ). Relative percentage to the wild-type at OD600 1.8. The purple area of the 3-oxo-C6AHL signals from TLC was calculated using the ImageJ program. Values are means ± standard deviation (SD) of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. wild-type. (C) β-Galactosidase activity reporting eanI expression in PA13L, ∆hfq mutant (−), and complementation strain (+; pLAFR3::hfq ). Values are means ± standard deviation (SD) of three independent experiments. ***p < 0.01 vs. PA13L.
Fig. 8. Proposed model of carotenoid production for the previously reported regulatory network HfqArcZ → RpoS Ͱ ClpXP and that identified here in which Hfq-controlled quorum signalling de-represses EanR to activate RpoS, thereby initiating carotenoid production. Carotenoid production confers tolerance to toxoflavin and UV radiation.
Supporting information figure legends
Supplementary Fig. S1. Recombinant plasmids for rearrangement of the carotenoid genes responsible for synthesising phytoene, lycopene, and β-carotene. The SOE by PCR products were first cloned into pGEM-T Easy, digested with Xhol and Sacl, and ligated into the corresponding position of pBBR1MCS5. Open triangles indicate the lacZ RBS. The SacI site, which is dotted and parenthesised, was from the pGEM-T Easy vector.
Supplementary Fig. S2. Confirmation of transcriptional units in the reassembled crtEB , crtEBI , and crtEBIY operons by RT-PCR. RT-PCR products were confirmed by Southern hybridisation. Black arrows indicate the extension and transcription directions of the crtEB ,crtEBI , andcrtEBIY operons on plasmids pYS71 (A), pYS69 (B), and pYS76 (C), respectively. Arrows below the open arrows represent the products of RT reactions. The short thick bars below the RT arrow indicate the PCR products from the corresponding RT reactions. The expected sizes of the PCR products are indicated below the labels. Agarose gel analysis (upper panel) and Southern analysis (lower panel) of the RT-PCR products of the crtEB ,crtEBI , andcrtEBIY operons. Southern hybridisation was performed using thecrtEBIY operon region (2.2, 3, and 5 kb XhoI–SacI fragments of pYS71, pYS69, and pYS76, respectively) as probes. Lanes 1–3, 4–6, and 7–9 correspond to the products of PCR1, PCR2, and PCR3, respectively. Lanes 1, 4, and 7: PCR products from the DNA template as positive controls; lanes 2, 5, and 8: PCR products from the RNA template as negative controls; and lanes 3, 6, and 9: RT-PCR products.
Supplementary Fig. S3. Carotenoid production confers UV radiation tolerance to P. ananatis. The wild-type andcrtE::pCOK184 mutant harbouring pCOK218 exhibited greater UV radiation tolerance than the crtE mutant at wavelengths of 320–400 nm for 20 s; however, the crtE::pCOK184 mutant showed lower UV radiation tolerance than the wild-type.
Supplementary Fig. S4. QS system of P . ananatisPA13. (A) Genetic map of the eanR and eanI loci of the QS system in P . ananatis PA13 and mutant generation. The putative lux box is upstream of eanR , for comparison, theesaR lux box of P . stewartii subsp.stewartii and lux box of Vibrio fischeri are presented. Campbell insertion and non-polar deletion mutants were generated to determine if eanR regulates the expression ofeanI ; a) PA13L, non-polar deletion of lacZY genes from wild-type PA13 used in the β-galactosidase assays as the wild-type; b)eanI ::pCOK153 (pVIK112 carrying truncated eanI at both ends); c) ∆eanI , non-polar deletion of eanI ; d) ∆eanR , non-polar deletion of eanR ; and e) ∆eanR eanI ::pCOK153. (B) Characterisation and quantification of AHL signals of the PA13 derivatives: (a) PA13L; (b) eanI ::pCOK153; (c) ∆eanI mutant; (d) ∆eanR mutant; and (e) ∆eanR eanI ::pCOK153 mutant. Ethyl acetate extracts were applied to C18 reversed-phase thin layer chromatography (TLC) plates. AHL signals were visualised with the C . violaceumCV026 biosensor, and synthetic C6-HSL and 3-oxo-C6-HSL were used as AHL standards (s). (C) β-Galactosidase activity reporting eanIexpression.
Supplementary Fig. S5. Strategy for generating recombinant carotenoid genes responsible for synthesising phytoene, lycopene, and β-carotene. PCR products with their overlapping regions aligned and the final rearrangement products are shown. The SOE by PCR products AD, AF, and AH are shown. In each case, the overlapping region between the primers, and the priming region in which each primer recognises its template, was designed to have the ribosome binding sequence (RBS) of each gene. The XhoI recognition sequence and lacZ RBS were introduced at the beginning of SOE-AB products. Dotted arrows indicate the rearrangements of carotenoid genes.
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