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.