INTRODUCTION
Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding (Song et al ., 2013; Ram et al ., 2020). Carotenoids are produced by plants and microorganisms including algae, fungi, yeast, and bacteria, but animals must obtain carotenoids from dietary sources. Interestingly, aphids, which are capable of synthesising carotenoids, are reported by later gene transfer from fungi (Moran and Jarvik, 2010).
A number of carotenoid-producing bacteria have been identified (Lorquinet al ., 1997; Dufossé et al ., 2005; Sodkova et al ., 2005; Sajilata et al ., 2008; Fasano et al ., 2014; Virtamoet al ., 2014; Lu et al ., 2017; Fidan and Zhan, 2019; Ramet al ., 2020). Carotenoids are highly hydrophobic, restricted to essential parts of the complex membrane and cell wall in bacteria, and mainly responsible for enhancing various functions related to the cell membrane and walls (Kirti et al ., 2014, Lutnaes et al ., 2004, Vila et al ., 2019). Carotenoids enhance various membrane functions, including physical strength, fluidity, cell wall rigidity, and lipid peroxidation. Several functions are closely related to the habitats of bacteria; in particular, the carotenoids of bacterial species living in low- or high-temperature environments are used to control the membrane fluid, while those of bacteria continuously exposed to UV radiation increase tolerance to UV (Kunisawa and Stanier, 1958; Mathews and Sistrom, 1959; Stanier, 1959; Mathew and Sistrom, 1960; Dundas and Larsen, 1963; Mostofian et al ., 2020). In addition, carotenoids aid bacteria in combating stress related to oxidation, salt, and desiccation (Oren, 2009; Tian and Hua, 2010). When bacteria are placed in a stressful environment, carotenoid production increases to protect against particular stressors, such as temperature, salt, light, and acidity (Paliwal et al ., 2017; Ram et al ., 2019). This is consistent with the fact that bacterial carotenoid production is closely related to habitat characteristics.
Pantoea ananatis is considered as an emerging pathogen based on the increasing number of reports of diseases occurring in a wide range of economically important agricultural crops worldwide. This pathogen can also infect humans and numerous insects (Coutinho and Venter, 2009; Dutta et al ., 2016; Weller-Stuart et al ., 2017) and cause bacteremia infection (De Baere et al ., 2004). P. ananatisPA13 causes plant diseases such as rice grain rot, sheath rot, and onion center rot disease in Korea (Choi et al ., 2012a; Choi et al ., 2012b; Kim and Choi, 2012). This pathogen is a potential threat to stable rice production, in particular during the growing season, when the weather is hot and humid.
Quorum sensing (QS) is bacterial cell-to-cell communication with extracellular signalling molecules called autoinducers that are present in the environment in proportion to cell density (Platt and Fuqua, 2010). This system facilitates community coordination of gene expression and benefits group behaviours. QS of P . ananatis, which uses EanRI homologous to P . stewartii subsp.stewartii EsaRI, has revealed that EanR negatively regulates self-expression and EPS production, but not eanI expression (von Bodman and Farrand, 1995; Minogue et al ., 2005; Morohoshiet al ., 2007; Lee, 2015). In P . ananatis , 3-oxo-hexanoyl homoserine lactone (3-oxo-C6AHL) and hexanoyl homoserine lactone (C6AHL) signals are generated by EanI and secreted extracellularly. AHL signals bind EanR, an AHL receptor; this interaction de-represses the EanR negative regulator (Morohoshi et al ., 2007).
The RNA chaperone Hfq and sRNAs are important regulators of virulence inP. ananatis (Kang, 2017; Shin et al ., 2019). Hfq, a ring-shaped hexameric RNA binding protein, has many important physiological roles that are mediated by interaction with Hfq-dependent small RNAs (sRNAs) in bacteria (Brennan and Link, 2007). Hfq was first reported in Escherichia coli as a host factor important in the replication of bacteriophage Qβ (Muffler et al. , 1996). Hfq regulates the stress response protein RpoS, which controls many stress response genes (Brown and Elliott, 1996; Mandin and Gottesman, 2010; Hwang et al ., 2011); it also regulates virulence in several pathogenic bacteria (Sittka et al ., 2007; Chao and Vogel, 2010; Zeng et al ., 2013). In addition, it modulates a wide range of physiological responses in bacteria. The hfq deletion mutant exhibits several different phenotypes (Figueroa-Bossi et al ., 2006). The Hfq protein interacts with A/U-rich regions of untranslated sRNAs of 50–250 nucleotides with tree stem-loop sequence motifs (Lorenzet al ., 2010) and assists with sRNA base pairing with target mRNA (Beisel and Storz, 2010) and the regulation of gene expression (Vogel and Wagner, 2007; Fröhlich and Vogel, 2009; Bardill and Hammer, 2012). Hfq is required for the functioning of several regulatory sRNAs, including OxyS and RyhB (Storz et al ., 2004; Majdalani et al ., 2005; Aiba, 2007; Gaida et al ., 2013). sRNAs act as activators or repressors of protein translation through complementary base pairing with mRNA in response to change in environmental conditions (Gottesman et al ., 2006; Waters and Storz, 2009; Beisel and Storz, 2010). Several sRNAs regulate RpoS, including ArcZ. ArcZ (also called RyhA and SraH) binds Hfq and positively regulates regulatory RNA, which controls the translation of RpoS (Repila et al ., 2003). ArcZ also regulates virulence, exopolysaccharide (EPS) production, motility, and the hypersensitive response (HR) in bacterial plant pathogens (Papenfort et al ., 2009; Soper et al ., 2010; Baket al ., 2014; Zeng and Sundin, 2014; Schachterle and Sundin, 2019).
Bacteria are surprisingly rich producers of carotenoids. However, bacteria with a low carotenoid content are unsuitable for commercial use. Production of plant-based carotenoids in bacteria is easier than in eukaryotic organisms such as yeasts, fungi, and plants (Ram et al ., 2020). Previously, biosynthesis of carotenoids has relied on bacterial carotenoid genes and DNA recombination techniques. Because these methods depend on restriction sites, generating recombinant DNA fragments and rearranging multiple carotenoid genes is problematic. The technique of splicing by overlap extension by polymerase chain reaction (SOE by PCR) using asymmetric amplification was first developed for introducing mutations into the centre of a PCR fragment (llis et al ., 1986; Higuchi et al ., 1988; Ho et al ., 1989), making site-directed mutagenesis more flexible. Horton et al . (1989) modified SOE by PCR to allow DNA segments from two different genes to be spliced together by overlap extension. SOE has been applied to enhance site-directed mutagenesis (Xiao et al ., 2007; Duan et al ., 2013; Hussain and Chong, 2016), generation of nonpolar, markerless deletions in bacteria (Merritt et al ., 2007; Kim et al ., 2013; Xu et al ., 2013), multiple-site fragment deletion (Zenget al ., 2017), and generation of hybrid proteins of immunological interest (Warrens et al ., 1997).
We reassembled carotenoid genes (crtE , crtB , crtI , and crtY ) of P. ananatis using splicing by overlap extension (SOE) to enable production of phytoene, lycopene, and β-carotene in Escherichia coli . We found that carotenoids were responsible for toxoflavin tolerance in P . ananatis . We confirmed that carotenoid production in P . ananatis is dependent on RpoS, which is regulated positively by Hfq/ArcZ and negatively by ClpP, similar to an important regulatory network ofE . coli (HfqArcZ → RpoS Ͱ ClpXP). We also showed that Hfq-controlled quorum signalling de-represses EanR to activate RpoS, thereby initiating carotenoid production.