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 ofcrtE –X –Y –I –B and Z ; and
that for P . agglomerans ofcrtE –idi –crtX –Y –I –B 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::crtE–B) producing phytoene
(retention time 2 min, 280 nm); b, E. coliDH5α/pYS69(pBBR1MCS5::crtE–B–I) producing
lycopene (retention time 11 min, 470 nm); and c, E. coliDH5α/pYS76(pBBR1MCS5::crtE–B–I–Y)
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 crtE –B , crtE –B –I ,
and crtE –B –I –Y operons by RT-PCR. RT-PCR
products were confirmed by Southern hybridisation. Black arrows indicate
the extension and transcription directions of the crtE –B ,crtE –B –I , andcrtE –B –I –Y 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 crtE –B ,crtE –B –I , andcrtE –B –I –Y operons. Southern
hybridisation was performed using thecrtE –B –I –Y 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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