DISCUSSION
Pantoea ananatis is an emerging plant pathogen that causes severe loss of many crops and trees, such as maize, onion, rice, and Eucalyptus, worldwide (Coutinho and Venter, 2009; Weller-Stuart et al ., 2017). This bacterium has also been associated with insects and humans. It is considered as a pathogen that is being revised because of its virulence in a wide variety of plant hosts and saprophytic ability in diverse ecological niches (Coutinho and Venter, 2009). In this study, we focused on the carotenoid biosynthesis gene cluster of P. ananatis and performed gene reassembly for carotenoid production. Here, we investigated the ecological and physiological functions of regulatory mechanisms of the carotenoid production of P. ananatis .
Much effort has focused on the biosynthesis of carotenoids using bacterial carotenoid genes (Misawa et al ., 1995; Guerinot, 2000; Lee et al ., 2003; Mijts and Schmidt-Dannert, 2003). Techniques based on recombining DNA sequences rely on restriction sites, so the primer must contain the introduced restriction site, which should not be in the centre of the fragment (llis et al ., 1986; Higuchiet al ., 1988; Ho et al ., 1989; Horton et al .,1989). Moreover, if multiple cloning vectors are to be used, plasmid incompatibility is also a limiting factor. Here, we applied a SOE by PCR technique to recombine DNA sequences without relying on restriction sites. In this report, we describe the reassembly of the genes encoding bacterial carotenoid biosynthetic proteins as crtE‒B, crtE‒B‒I , or crtE‒B‒I‒Y for the synthesis of phytoene, lycopene, or β‑carotene, respectively. E . coli expressing crtE‒B, crtE‒B‒I , or crtE‒B‒I‒Y produced phytoene, lycopene, or β-carotene, respectively. Zeaxanthin biosynthesis was enabled by the addition of crtZ , but gene recombination failed despite numerous attempts. It seems that the likelihood of success decreases with increasing number of genes to be recombined.
In practice, simply introducing lacZ ribosomal binding sequences (RBSs) at the beginning of the SOE-AB product (PlaccrtE ) enables carotenoid biosynthesis. CrtE catalyses the synthesis of GGPP, an early intermediate of carotenoid biosynthesis. We did not test whether the absolute amount of carotenoids increases as the level of GGPP increases in vivo .
We used a DNA template from the rice pathogenic bacterium P .ananatis in SOE by PCR, which is controllable and independent of restriction sequences, for carotenoid gene reassembly. Pantoea agglomerans , which causes palea browning of rice, is a genetically close species to P . ananatis with which it shares a biological niche. The organisation of the carotenoid biosynthetic gene clusters of the two strains is identical, except idi . Interestingly, P . agglomerans has an idi gene between crtE and crtX , which distinguishes it fromP . ananatis , suggesting that idi could be used to distinguish genetically similar bacteria.
SOE is a novel PCR-mediated recombinant DNA technology that does not rely on restriction sites, so its coverage is considerably wider than standard restriction enzyme-based methods for gene recombination. This enables finer control over recombination for genetic engineering. In addition, the sequence of the overlap region is determined by primer design, allowing simultaneous non-polar mutagenesis, site-directed mutagenesis, and recombination. In this study, we applied this technically simple and rapid recombinant DNA technique to the biosynthesis of three carotenoids. The technique will likely be suitable for recombination of multiple genes.
In bacteria, carotenoids are closely related to the conditions of the surrounding environment. We found that the UV radiation tolerance ofP . ananatis was due to the carotenoids they produce. These results are consistent with those regarding P . stewartiisubsp. stewartii (Mohammadi et al ., 2012). Considering the plant environment (particularly rice) in which P . ananatislives, UV radiation tolerance is advantageous for survival. Interestingly, these carotenoids are unique in that they also makeP . ananatis tolerant to toxoflavin. Thus, tolerance to toxoflavin via carotenoid production can be considered a survival strategy of P . ananatis . Bacteria that produce carotenoids have advantages for overcoming environmental stresses, such as UV radiation, salt, and low temperatures. Pantoea ananatis andB . glumae share the same rice environment and are the first reported cases of the production and use of carotenoids to overcome toxoflavin.
We found that QS and Hfq are directly or indirectly involved in regulating carotenoid production in P. ananatis PA13. QS regulates an extensive range of functions, including bioluminescence, virulence, biofilm formation, DNA exchange, and sporulation in bacteria (Fuqua et al ., 1996, Waters and Bassler, 2005). Hfq is a global RNA chaperone that interacts with sRNAs of diverse functions; it also regulates of virulence and environmental stress in many plant and animal bacterial pathogens (Ding et al ., 2004; Chao and Vogel, 2010; Zeng et al ., 2013; Shin et al ., 2019). The hfqmutant in Erwinia amylovora Ea1189 reduces virulence, amylovoran EPS production, biofilm formation, motility, and positive regulation of the type III secretion system (Zeng et al ., 2013). InPectobacterium carotovorum , the hfq mutant exhibits defects in motility, biofilm formation, sedimentation, and virulence (Wang et al ., 2018). Hfq is also an important regulator of virulence, motility, and biofilm formation in P. ananatis LMG2665 (Shin et al ., 2019). We found that Hfq regulates the expression of eanI encoding the QS signal synthase, which was confirmed byeanI expression and QS signal productivity assays. These results are consistent with the finding that Hfq regulates QS signal production directly via interactions with the AHL receptor ExpR inSinorhizobium meliloti (Gao et al ., 2015). QS systems integrate other global regulators, including noncoding sRNAs. This network is activated through the binding of Hfq and Hfq-dependent sRNA and controls gene expression via post-transcription regulation (Storzet al ., 2005). There are several reports that the Hfq-dependent sRNAs Qrr1–4 and RsmY interact with Hfq to directly and indirectly control QS targets in Vibrio cholerae and Pseudomonas aeruginosa (Lenz et al ., 2004; Kay et al ., 2006). Shinet al . (2019) suggested that the putative Hfq-dependent sRNAs pPAR237 and pPAR238 are involved in regulating QS by activating EanI without genetic analyses. Further studies are needed to identify the sRNAs in P. ananatis . It was previously reported that EanR mediated QS regulation by de-repression as in P. stewartii (von Bodman and Farrand, 1995; Morohoshi et al ., 2007). In P. ananatis , EanR represses the ean box (lux box-like sequences) in the upstream region of eanR , and adding AHL promoted dose-dependent de-repression (Morohoshi et al ., 2007). This EanR-mediated QS regulation was similar to that of the close homolog EsaR in P. stewartii (Minogue et al ., 2005). Overall, we found that QS signal production in P . ananatiswas delayed in the absence of Hfq, since EanR negatively regulates RpoS. Expression of RpoS is entirely dependent on bacterial growth. Using EanR, P . ananatis must inhibit RpoS expression before reaching stationary phase, at which point EanR is removed to initiate expression of RpoS. Hfq is responsible for determining the timing of the Hfq-mediated increase in eanI expression to produce full QS signals. The resulting QS signals de-repress EanR, followed by Hfq to express RpoS, which turns on carotenoid biosynthesis.
We found that RpoS regulates carotenoid biosynthesis under the control of Hfq, QS, and ClpP. The regulatory networks of HfqArcZ → RpoS Ͱ ClpXP for carotenoid production are similar to those of E . coli . Here, we elucidated a regulatory network of carotenoid production involving Hfq-dependent QS‒RpoS in P . ananatis . Hfq regulates full production of QS signals, thereby de-repressing the EanR negative regulator to initiate RpoS expression (Fig. 8).