Cloning of SOE fragments and RT-PCR analysis
Each SOE product was TA-cloned into pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced to confirm the presence of the DNA sequences (Macorgen Inc., Daejeon, South Korea). Next, clones containingcrtEB , crtEBI , or crtEBIY were digested with Xhol and Sacl and ligated into the corresponding positions of pBBR1MCS5 (Kovach et al ., 1995), generating pYS71, pYS69, or pYS76, respectively (Supplementary Fig. S1).
We used RT-PCR to determine if the reassembled crtEB ,crtEBI , and crtEBIY clones on the plasmids pYS71, pYS69, and pYS76 are transcribed as a single transcript. We used three sets of primers to amplify crtEB , BI , andIY . RT-PCR followed by Southern hybridisation indicated that the reassembled crtEB ,crtEBI , andcrtEBIY clones on the plasmids pYS71, pYS69, and pYS76 are transcribed as single transcripts (Supplementary Fig. S2).
Carotenoid production in E. coli
To determine if E . coli DH5α transformed with pYS71/pSRKGm::crtEB , pYS69/pSRKGm::crtEBI , or pYS76/pSRKGm::crtEBIY produces phytoene, lycopene, or β-carotene, respectively, we performed high-performance liquid chromatography (HPLC). The results revealed thatE . coli DH5α/pYS71/pSRKGm::crtEB produced colourless phytoene, as confirmed by the standard peak at the same retention time (Fig. 3A and D); E . coliDH5α/pYS69/pSRKGm::crtEBI produced magenta lycopene, as confirmed by the standard peak at the same retention time (Fig. 3B, D); and E . coliDH5α/pYS76/pSRKGm::crtEBIY produced orange β-carotene, as confirmed by the standard peak at the same retention time (Fig. 3C, D). SOE enabled reassembly of multiple carotenoid synthetic genes and the production of carotenoids inE . coli .
Carotenoid confers P. ananatis with tolerance to toxoflavin and UV radiation
Toxoflavin is a phytotoxin produced by B . glumae , a rice-grain pathogen that shares rice environments with P .ananatis and has antibacterial properties. To determine if carotenoid production in P . ananatis is responsible for tolerance to toxoflavin and UV radiation, we generated a polarisedcrtE ::pCOK184 mutant by Campbell insertion (Fig. 4A). Complementation plasmid pCOK218 was also generated by cloning the carotenoid biosynthetic genes crtEZ into pBBR1MCS5 (Fig. 4A), which recovered the carotenoid deficiency in thecrtE ::pCOK184 mutant (Fig. 4B). The wild-type is sensitive to toxoflavin concentrations > 20 µg mL−1(Fig. 4B). The crtE ::pCOK184 mutant exhibited lower tolerance than the wild-type to 20 µg mL−1 toxoflavin; however, the wild-type and complementation strain (+) showed greater tolerance than the crtE mutant (Fig. 4B). These results were consistent with those for UV radiation tolerance, but the survival of thecrtE ::pCOK184 mutant was approximately 100 times lower than that of the wild-type (Supplementary Fig. S3).
Carotenoid production is dependent on RpoS, which is regulated positively by Hfq/ArcZ and negatively by ClpXP in P. ananatis
Figure 5A shows the HfqArcZ → RpoS Ͱ ClpXP regulatory networks of E . coli . In E . coli , the stationary sigma factor RpoS is regulated positively by Hfq and its cognate sRNA ArcZ. RpoS levels are kept low by constitutive degradation of the ClpXP protease until stationary phase (Raju et al ., 2012). RpoS-dependent carotenoid production has been previously reported inP . agglomerans (Becker-Hapaka et al ., 1997). To determine whether this regulation also occurs in P .ananatis , we constructed non-polar mutants of the rpoS ,hfq , arcZ , and clpP genes and generated complementation strains for the corresponding gene mutants. The colonies of ∆rpoS and ∆hfq mutants were white, and neither produced carotenoids (Fig. 5B); however, colonies of complementation strains (+) carrying pCOK312 and pCOK335, respectively, were orange and produced carotenoids. Colonies of the ∆arcZ mutant were faint orange and exhibited a slight reduction in carotenoid production (Fig. 5B), indicating involvement in carotenoid production. Colonies of the ∆clpP mutant were dark orange and exhibited an approximately two-fold increase in carotenoid production, indicating negative carotenoid regulation via RpoS inhibition (Fig. 5B). Complementation strains (+) of ∆arcZ and ∆clpP mutants carrying pBS28 and pOR78, respectively, produced amounts of carotenoids similar to that of the wild-type. These results suggest that carotenoid production ofP . ananatis is dependent on RpoS, which is regulated positively by Hfq/ArcZ and negatively by ClpP, similar to an important regulatory network of E . coli (HfqArcZ → RpoS Ͱ ClpXP).
EanR negatively regulates carotenoid production in P. ananatis
A previous report examined EanR de-repression in the QS system ofP . ananatis , which causes centre rot disease in onion (Morohoshi et al ., 2007). QS of P . ananatis PA13 is also similar to that of P . stewartii (Minogue et al ., 2005). Supplementary Fig. S4 shows the QS system of P .ananatis PA13. The eanR and eanI genes are transcribed in the opposite direction, and the lux box is at theeanR gene promoter region (Supplementary Fig. S4A). To determine if eanI expression is under the control of EanR, we constructed alacZY integration of eanI ::pCOK153 (i.e., pVIK112 carryingeanI truncated at both ends) in PA13L and PA13L∆eanRmutant backgrounds using Campbell insertion (Supplementary Fig. S4A). QS signal production of the mutants was confirmed using thin-layer chromatography (TLC) and a Chromobacterium indicator strain. TheeanI mutant did not produce QS signals, whereas the eanRmutant did (Supplementary Fig. S4B). The expression of eanI was not decreased in the ∆eanR mutant background or increased by the addition of 3-oxo-C6AHL or C6AHL (Supplementary Fig. S4C). These data indicate that the expression of eanI is not under the control of EanR.
We performed functional phenotypic de-repression of EanR using ∆eanI , ∆eanR , and ∆eanI‒R mutants of P .ananatis PA13. The ∆eanI mutant exhibited no production of QS signals or carotenoids; however, ∆eanR and ∆eanI‒Rmutants produced carotenoids, indicating that EanR negatively regulates carotenoid production by the binding of AHLs to EanR (Fig. 6A−C). Carotenoid production of the ∆eanI‒R mutant was abolished by transformation with pCOK199 (pBBR1MCS5::PlaceanR ), confirming that EanR negatively regulates carotenoid production (Fig. 6B, C).
EanR negatively regulates carotenoid production via inhibition ofrpoS in P. ananatis
To determine if rpoS expression is regulated by EanR, we constructed a lacZY integration of rpoS ::pYS88 (pVIK112 carrying rpoS truncated at both ends) in PA13L, PA13L∆eanI , PA13L∆eanR , and PA13L∆eanI‒R mutant backgrounds using Campbell insertion. The expression of rpoSdecreased significantly in the ∆eanI mutant background.rpoS expression increased in the ∆eanR and ∆eanI‒Rmutant backgrounds (Fig. 6D); rpoS expression decreased in the presence of EanR. These results indicate that EanR negatively regulatesrpoS expression and QS signals de-repress EanR. Although the putative lux box suggests that EanR binds to the promoter region of rpoS (Fig. 6E), there is currently no direct evidence for this. We analysed the candidate lux box(s) in the crtEXYIBgene cluster or the promoter region of the crtZ gene, but did not find it.