Whole genome analysis of Enterobacter cloacae Rs-2 and screening of genes related to plant-growth promotion

The genus Enterobacter is widely recognized for its biotechnology potential in improving soil environment and crop growth promotion. To further explore these biotechnological potentials, we sequenced and analyzed the whole genome of Enterobacter cloacae Rs-2. The analysis showed that the total length of the Rs-2 genome was 6,965,070,514 bp, and GC content was 55.80%; the annotation results of GO and COG databases showed that the genome contains a variety of growth-promoting genes, such as iscU, glnA, glnB (nitrogen fixation); iucABCD (siderophore synthesis) and fepA, fcuA, fhuA, and pfeA, etc. (siderophore transport); ipdC (secreted IAA) and gcd, pqqBCDEF (dissolved phosphorus), etc. No pathogenic factors such as virulence genes were found. The application of Rs-2 as a soil inoculant in pot experiments showed great potential for growth promotion. This study proved the plant growth-promoting ability of Rs-2 at the molecular level through genetic screening and analysis, which provided guidance for the further improvement of the strain and laid a foundation for its application in agricultural production.


Introduction
The process of global agricultural production is often accompanied by the application of chemical fertilizers. The unreasonable use of chemical fertilizers in the farmland often leads to fertilizers in the soil being poorly utilized, effective nutrients will be adsorbed or fixed in the soil, and it will be difficult to be uptaken and utilized by plants. In addition, the accumulation of heavy metals and the newly formed compounds from excessive application of chemical fertilizer will cause soil salinization and pollution as well as groundwater pollution along with leaching and transpiration. These environmental problems will not only reduce food production but also be harmful to human health. Much literature have shown that plant growth-promoting rhizobacteria (PGPR) can simultaneously promote host plant growth in multiple ways, including nitrogen fixation (Hoffman et al. 2014), siderophore secretion (Chaiharn et al. 2009), insoluble phosphates (IP) dissolution (Li et al. 2016), plant growth hormone secretion (Soni et al. 2021;Kang et al. 2016), etc., and can also enhance the survival ability of plants under salt stress conditions by inducing plants to establish resistance or tolerance mechanisms (Vimal et al. 2017). PGPR can simultaneously promote plant growth in multiple ways, so it will be necessary to explore the synthetic pathway and regulation mechanism of biomass-promoting substances.
With the development of molecular biology technology, whole genome sequencing provides favorable conditions to analyze the physiological and ecological functions of PGPR. Soni et al. (2021) revealed the genetic characteristics of P. polymyxa HK4 in phosphorus solubility, siderophore production, etc. through draft genome analysis, and verified its biological control and plant growth promotion abilities. Xu et al. (2022) identified genes related to plant growth in R. aceris ZF458 through genome annotation and comparative genomics, which laid the foundation for the analysis of plant growth-promoting genes. Functional genes such as phosphorus dissolution, nitrogen fixation, and siderophore production, etc., can be excavated by molecular biology techniques to explain the main growthpromoting properties, so as to deepen the understanding of the growth-promoting and disease-preventing life activities of strains at the molecular level (Cleton-Jansen et al. 1991;Kang et al. 2016;Silva et al. 2020;Straub et al. 2013). Therefore, mining functional genes are very important and valuable to strengthen the research on the growth-promoting characteristics of strains, supplement the metabolism of strains, and improve the application efficiency of PGPR in plants.
Enterobacter cloacae Rs-2 was isolated from pepper rhizosphere soil of saline-alkali land. Previous experiments have shown that Rs-2 has good biological control and plant growth promotion properties (Wu et al. 2012;Li et al. 2021). However, the metabolic pathways and mechanisms of action for these functions are still unclear. We hypothesize that the biological control and plant growth promotion properties of strain Rs-2 were linked with their key growth-promoting genes. Therefore, the genome of Rs-2 was sequenced and the purpose of this study was to reveal and verify its various gene functions involving growth-promoting factors (nitrogen fixation, phosphorus solubilization, production and transport of siderophore, etc.), annotating the whole genome of Rs-2, and determining its growth-promoting properties and abilities in plants. These data will provide insight into the mechanisms of plant growth promotion.

Sample collection, culture conditions, and DNA extraction
The E. cloacae Rs-2 was isolated from the pepper rhizosphere (5-25 cm) soil of cotton saline-alkali land in Xinjiang, China. The complete sequence of Rs-2 has been uploaded to NCBI under the accession number PRJNA643379.
The strain Rs-2 was stored in glycerol (V/V = 50%) at − 20 ℃ and for the experiment was activated in LB liquid medium (10 g/l tryptone, 5 g/l yeast dip powder, 10 g/l NaCl, and pH 7.0) at 30 ℃ and 180 rpm for 12 h, the inoculum of bacteria is 1% (V/V).
DNA extraction: 1 ml of the above Rs-2 culture solution was placed in a 2-ml sterile enzyme-free centrifuge tube, centrifuged at 4 °C and 1000 rpm for 3-5 min, and resuspend the pellet in PBS (pH = 7.0) buffer. The process is repeated twice. The obtained cells were frozen in liquid nitrogen for 2 h and then quickly transferred to a -80 °C freezer for storage. After that, the total DNA was extracted by the CTAB method (Porebski et al. 1997) and stored at -20 °C.

Whole genome sequencing, assembly, and annotation
The Rs-2 whole genome was sequenced using Illumina NovaSeq and PacBio Sequel sequencing platforms. Construction of different insert libraries used whole genome shotgun (WGS) strategy. The sequencing work was entrusted to Beijing Biomaike Biotechnology Co., Ltd. Assessment of sequencing data quality was done using FastQC software and corrected by SOAPec (Frey et al. 2014), and then the third-generation sequencing data were assembled using HGAP (Chen et al. 2016) and CANU (Koren et al. 2017) software to obtain contig sequences. The gene sequences obtained by whole-genome sequencing of Rs-2 were compared with general databases (GO and COG) by BLAST to obtain gene function annotation results. GO functions were annotated with BLAST2GO software, and GO classification of Rs-2-encoding genes was performed according to molecular function (MF), biological process (BP), and cellular component (CC). In addition, through the COG analysis, the functions of the sequences and metabolic pathways were annotated and predicted.

Identification of Enterobacter strains
The Rs-2 was identified by the method of 16S rRNA gene sequence comparison. The genomic DNA of Rs-2 was extracted, and then the conserved region 16S rRNA gene fragment of the strain was amplified by PCR using universal primers 27F (AGA GTT TGA TCC TGG CTC AG) and 1492R (CGG TTA CCT TGT TAC GAC TT) (Li et al. 2007). The obtained 16S rRNA gene sequence was compared with the sequence of high similarity in GenBank by the BLAST program (Yoon et al. 2017). Phylogenetic trees were constructed using Molecular evolutionary genetics analysis (MEGA) software, confirmed by the maximum likelihood (Felsenstein 1981) method, and 1000 replicates were subjected to bootstrap analysis (Felsenstein 1985).

Bacterial growth curve
A 1% (V/V) Rs-2 was inoculated into a 250 ml Erlenmeyer flask containing 50 ml LB liquid medium and cultured continuously for 7 days at 30 °C and 180 rpm. The change in pH of the culture medium was measured during the period, and the concentration of viable bacteria was measured by the plate gradient dilution method. The optical density (OD) of the culture solution was measured with a spectrophotometer at the UV-visible wavelength of 600 nm, and ultrapure water was used as a control, which was recorded as OD 600 .

Nitrogen fixation
The nitrogen fixation ability of Rs-2 was qualitatively determined by inoculating it into Ashby's medium. The light blue halo formed around the colony indicates that the bacteria can fix nitrogen in the atmosphere.

Siderophore production
The siderophore secretion capacity of Rs-2 was determined by CAS qualitative and quantitative methods. The ability of Rs-2 to secrete siderophore was qualitatively tested by the CAS plate assay (Lambrese et al. 2018). The positive reaction was determined by the color of the CAS solid medium changing from blue to lighter or transparent, and the halo formed around the colony was recorded.
Inoculate 1% (V/V) Rs-2 bacterial seed liquid into 250-ml Erlenmeyer flasks containing 50 ml MSA (iron-free) and Fe-MSA (100 μmol/l FeSO 4 ·7H 2 O) liquid medium, respectively. The culture medium was continuously cultured for 7 days. After 2 ml of the sample was centrifuged, an equal volume of CAS detection solution was added to the supernatant and reacted in the dark for 1 h to obtain OD 630 (A). The OD 630 (Ar) was measured with ultrapure water as a control. The relative amount of siderophore secreted by Rs-2 was calculated by the formula (Ar−A)×100% Ar .

IAA production
Rs-2 was subcultured on sterile LB liquid media containing L-tryptophan (100 mg/l). The ability of Rs-2 to synthesize IAA was determined using the principle of Salkowski chromogenic (Glickmann and Dessaux 1995).

ACC deaminase activity
The determination of ACC deaminase (ACCD) activity was referenced to the method of Penrose et al. (Penrose and Glick 2003). The unit enzyme activity (U) and total protein mass were determined by the 2,4-dinitrophenylhydrazine chromogenic method and Coomassie brilliant blue G-250 method, respectively, and the ratio of the two (U/mg) was the ACCD activity. The experiment was repeated three times.

Phosphorus solubilization
The 1% (V/V) Rs-2 strain was inoculated into Pikovskaya (PVK) medium with Ca 3 (PO 4 ) 2 as the only phosphate, and the available phosphorus content was measured by molybdenum blue colorimetry (Chang and Yang 2009).
To characterize the IP-soluble metabolites of Rs-2, the species and concentrations of organic acids in PVK medium were determined by high-performance liquid chromatography (HPLC).
The quantitative HPLC analysis was performed on the Shimadzu LC equipped with the Shimadzu Wondasil C 18 column (4.6 × 200 mm). The mobile phase was 0.0075 mol/l H 2 SO 4 (100%), the flow rate of the mobile phase was 0.5 ml/min, the column temperature was 35 °C, the injection volume of the sample was 10 μl, and the UV detection wavelength was 210 nm.

Pot trials
The cells of Rs-2 were inoculated in a sterile 1.5 kg vermiculite: perlite = 3:1 mixed matrix, at a density of 10 8 CFU/g of the matrix, with 2 g of Ca 3 (PO 4 ) 2 applied per pot. The radish seeds were soaked in 75% ethanol solution for 5 min, then used a 0.2% HgCl 2 solution for surface sterilization for 10 min, and rinsed three times with sterilized water. Radish seeds were planted in pots at a depth of 2 cm, with 5 radish seeds per pot. There were 4 replicates for each potting treatment. Plants were grown in an artificial climate box (Shanghai Yiheng Scientific Instrument Co., Ltd.) with conditions (day and night temperature 28/25 °C, lighting for 12 h per day, relative humidity of 40%, and light intensity of 250 μmol/m 2 ), watering once a day to keep the substrate moist. A total of 30 ml of Rs-2 seed solution (OD 600 = 1.0) suspended in sterile water was inoculated on days 10 and 20, respectively. The effect of Rs-2 on radish seeds was recorded after 30 days of sowing, and the fresh weight, dry weight, number of leaves, root length, chlorophyll, carotenoid C, soil available phosphorus, and total plant phosphorus content were detected, respectively.
The soil and impurities on the plants were washed with ultrapure water, and the fresh weight was weighed after absorbing the water with absorbent paper. Transfer to an oven at 80 ℃ for 24 h, and weigh when the mass no longer changes.
The content of chlorophyll and carotenoid C in leaves was determined by the Lichtenthaler method (Lichtenthaler and Wellburn 1983). A total of 0.2 g of leaves were weighed into a test tube and 10 ml of extract solution (acetone: ethanol = 1:1) was added, shaken, and stored overnight in the dark. When the leaf samples were bleached, the OD 470 , OD 663 , and OD 646 of the extract were measured using a UV spectrophotometer. The chlorophyll content in the leaves was calculated by the following formula: where V and M are the total volumes of extract (l) and the fresh weight of radish leaves (g), respectively.
After the shoots and roots of the radish plants are ground and dried, 0.1 g of the shoots and all the roots are put into the digestive tube, respectively, moistened with ultrapure water, and boiled with H 2 SO 4 -H 2 O 2 (the temperature should not exceed 320 °C) for 2 h until digestive juice is clear. After the digestion solution was cooled, the volume was adjusted to 50 ml, and the total phosphorus content of radish plants was determined by the vanadium molybdenum yellow colorimetric method.
The soil from the roots of the radish was selected for the experiment, dried in an oven at 80 ℃ to constant weight, and then passed through a 2-mm sieve after grinding. A 2.5 g of treated soil pellets were immersed in 50 ml NaHCO 3 leaching solution for 30 min at 25 °C and 180 rpm. After centrifugation, the available phosphorus content in the supernatant was determined by molybdenum blue colorimetry.

Resistance to antibiotics
Antibiotic susceptibility experiments were carried out on the isolated and purified E. cloacae Rs-2 by the Clinical and Laboratory Standards Institute (CLSI) disc diffusion methods. A 0.1 mL of the bacterial suspension with OD 600 = 0.8 was evenly spread on the LB agar medium, and then the antibacterial drug paper (Hangzhou Microbial Reagent Co., Ltd.) was attached to the surface of the medium. The interval between antibiotic discs is 24 mm, and the edge of the disc is not less than 15 mm from the edge of the medium. The diameter of the inhibition zone was determined after culturing at 30 °C for 24 h (R = bacteria can grow normally, indicating that Rs-2 is resistant to antibiotics (d ≤ 10 mm); I = the growth of Rs-2 was slightly restricted compared to R, indicating that Rs-2 is moderately sensitive to antibiotics (10 mm < d ≤ 20 mm); S = the growth of Rs-2 was severely restricted, indicating sensitivity to the antibiotic (d > 20 mm)). The test for each antibiotic was repeated three times.

Data analysis
One-way ANOVA was performed on experimental data using IBM SPSS Statistics software with Tukey, LSD, and Bonferroni multiple comparison methods as posttests.

General genome characteristics and strain identification of Rs-2
The automated annotation of the genome of Enterobacter cloacae Rs-2 by the GO and COG databases did not reveal any significant differences. The genome size was 6,965,070,514 bp, and the G + C content was 55.80% ( Fig. 1). The predicted coding genes number was 4226, and the size was 4,132,281 bp. Noncoding RNA predictions indicated 25 rRNAs and 83 tRNAs, and 102 other ncRNAs. Phylogenetic analysis showed that Enterobacter cloacae Rs-2 is grouped in a cluster with the strain Enterobacter cloacae ECL72 (CP077659.1) (Fig. 2). The results of the gene sequence searched by BLAST showed that the nucleotide homology of the Enterobacter cloacae Rs-2 strain was 99.28%, 99.28%, 99.21%, and 93.71% compared with Enterobacter cloacae ECL72, Enterobacter cloacae AR 0060, Enterobacter cloacae STN0717-60, and Enterobacter cloacae subsp. cloacae type strain ATCC 13047, respectively. The genome of Rs-2 exhibited the general characteristics described for Enterobacter genomes (Panigrahi et al. 2020;Naushad et al. 2019).
GO annotation results showed that Rs-2 gene function was mainly concentrated in biological process (BP), with 1762 genes related to metabolic processes, 1486 genes related to cellular processes, and 1398 genes related to single-organism processes, as well as 299 genes associated with response to stimulus (Fig. 3). The results of annotating different sequences into different COG categories showed that there are at most 522 genes enriched in R category (General function prediction only), such as the prediction of dehydrogenases, the prediction of nucleic-acid-binding proteins, etc. There were 22 types of genes encoding proteins, with a total of 4299 genes, of which 454 genes were responsible for carbohydrate transport and metabolism (G), 444 genes encoded amino acid transport and metabolism-related proteins (E), and 354 genes encoded transcription-related proteins (K), indicating that COG-annotated genes are mostly related to the basic functions of bacterial cells (Fig. 4). In addition, 64 genes related to the biosynthesis, transport, and metabolism of secondary metabolites, such as various organic acids involved in the synthesis of insoluble phosphorus dissolution. There are also some genes whose functions are unknown (S) and need to be further explored.

Pathogenicity and resistance
Sometimes E. cloacae were considered to be human opportunistic pathogens because they were prone to cause respiratory infections and other diseases and were resistant to antibiotics (Christie and Vogel 2000). E. cloacae subsp. cloacae type strain ATCC 13047 is a typical human opportunistic pathogen. Seven loci encoding fimbrial biosynthesis proteins and some other virulence factors were identified on the chromosome and plasmid pECL_A of ATCC 13047 (Ren et al. 2010), which were not found in Rs-2.

Fig. 1
Circular visualization of Rs-2 genomic maps. The first circle from the outside to the inside is the genome size unit, the unit is 5 kb; the second and third circles represent genes in the positive and negative strands of the genome, respectively, and COG functional classi-fications are represented by different colors; the fourth circle represents repeat sequences; the fifth circle is blue for tRNA, and purple for rRNA; the sixth circle is the GC content; the innermost circle is the GC-skew (G -C / G + C) value  In addition, the results of susceptibility testing of Rs-2 to 30 different antibiotics showed extensive antibiotic resistance (Table 1). The bacteria have the highest sensitivity to ciprofloxacin, ofloxacin, and norfloxacin but have strong resistance to 15 antibiotics such as clindamycin, furazolidone (ditrin), and Rs-2, and have moderate sensitivity to other 8 antibiotics such as chloramphenicol and cotrimoxazole.

Plant growth-promoting characteristics
The promotion of plant growth by PGPR mainly depends on a variety of mechanisms, such as (i) stimulating plant uptake of nutrients; (ii) secreting growth regulators and growth factors that are beneficial to plants; (iii) regulating the tolerance of plants to abiotic stresses. Rs-2 is a PGPR with multiple growth-promoting properties that have been investigated and described as follows:

Bacterial growth regularity
The Rs-2 was inoculated into LB liquid medium for continuous culture, and the viable bacterial density, OD 600 , and pH at different stages were determined (Fig. 5a). The results showed that Rs-2 was in logarithmic growth phase before 48 h, and the maximum viable bacterial density was 3.60 × 10 8 CFU/ml. The trend of the viable bacterial density curve and OD 600 curve is consistent. In addition, the pH of fermentation broth is increased with time, which is always alkaline (pH > 7). Table 1 Sensitivity of E. cloacae Rs-2 to 30 antibiotics R, bacteria can grow normally, indicating that Rs-2 is resistant to antibiotics (d ≤ 10 mm); I, the growth of Rs-2 was slightly restricted compared to R, indicating that Rs-2 is moderately sensitive to antibiotics (10 mm < d ≤ 20 mm); S, the growth of Rs-2 was severely restricted, indicating sensitivity to the antibiotic (d > 20 mm) When Rs-2 was cultured on NFB solid medium, the blue halo appeared (D = 2.95 ± 0.17 cm), suggesting that Rs-2 has the nitrogen-fixing ability (Fig. 5b). And the colony diameter (d) was 0.45 ± 0.06 cm, indicating the nitrogen fixation Index (NFI = D/d) was 6.56.

Siderophore production
Rs-2 formed a visible transparent halo on the CAS detection plate in Fig. 5d, with a diameter of 2.1 ± 0.1 cm, which indicated that the strain had the ability to produce a siderophore. In an iron-free MSA medium, Rs-2 produced a large amount of siderophore, and the relative content Nitrogen fixation qualitative test. c Siderophore production quantitative test. d Siderophore production qualitative test. e IAA production quantitative test. f ACCD activity quantitative test. g Phosphorus solubilization. h Organic acid secretion of siderophore was higher (> 90%) after 48 h (Fig. 5c). Interestingly, the relative content of siderophore detected in Fe-MSA medium is consistent with the trend of OD 600 curve, but the content of siderophore is much less than that of MSA (the maximum relative content is 31.63%, 7 days), while the OD 600 is higher than that of MSA, indicating that the cell density in iron-rich Fe-MSA medium is higher than that in MSA, which proves Rs-2 has an excellent ability to secret siderophore in Fe-deficient environments.

IAA production
The auxin IAA synthesized by PGPR with L-tryptophan (L-Trp) as the precursor can been absorbed on the surface of seeds and roots to stimulate the growth and reproduction of plant cells. As shown in Fig. 5e, Rs-2 was induced to synthesize IAA in LB medium with L-Trp as inducer, and the highest concentration on the 4th day was 196.74 ± 3.83 μg/ml. Although the cell density decreased after 48 h, the IAA concentration was still high, which would provide sufficient IAA for plant growth.

ACCD activity
The great contribution of IAA to the promotion of plant growth is also manifested in the induction of ACCD activity (Glick et al. 1998). Some studies have shown that PGPR reduce the stress effect of ethylene on plant growth by hydrolyzing ACC, the precursor of ethylene synthesis, to α-butanone acid and ammonia by producing ACCD (Glick et al. 1998;Prigent-Combaret et al. 2008). The highest ACCD activity of Rs-2 after induction was 4.69 ± 0.35 U/mg, as shown in Fig. 5f. Wu et al. (2012) indicated that inoculation cotton plants with Raoultella planticola resulted in a lower ethylene content than CK (bacteria-free) treatment, validating the ability of strains to produce ACCD.

Phosphorus solubilization
Phosphorus mainly exists in soil as insoluble inorganic salts, such as Ca 3 (PO 4 ) 2 , AlPO 4 , FePO 4 . Phosphorus quantification in PVK media inoculated with Rs-2 showed opposite changes in soluble P and pH (Fig. 5g). The maximum solubilized P of Rs-2 was 430.73 ± 13.02 mg/l (12 h) with pH decreased from 7.91 to 4.55. The soluble phosphorus concentration remained stable after the 3rd day, indicating that the absorbed and immobilized phosphorus was in equilibrium with the dissolved and released phosphorus. Various extracellular substances secreted by PGPR can effectively lower the pH of the surrounding environment, such as organic acids, which play a crucial role in the dissolution of IP. Organic acids released by Rs-2 in vitro during IP dissolution were detected by HPLC (Fig. 5h). The results showed that Enterobacter cloacae Rs-2 could secrete gluconic acid, acetic acid, lactic acid, and malic acid. The content of each organic acid was relatively low at 6 h, i.e., from 0.12 to 0.48 mg/l, and then the content of lactic acid and malic acid increased significantly, while the content of gluconic acid decreased. In addition, malic acid (0.91 ± 0.09 g/l), acetic acid (0.79 ± 0.13 g/l), and gluconic acid (0.41 ± 0.01 g/l) levels peaked on the third day. Furthermore, during the 5-day observation period, the content of lactic acid was highest than that of malic acid, acetic acid, and gluconic acid, with the highest concentration of 1.46 ± 0.10 g/l on the fourth day. Although gluconic acid is considered to be one of the main mechanisms of phosphorus dissolution, its content is lower than that of lactic acid. Therefore, we believe that lactic acid plays a crucial role in the process of IP dissolution by Rs-2.

Investigation of growth-promoting function genes
Nitrogen is an essential nutrient element for plant growth, but it exists mainly as nitrogen (N 2 ) in the atmosphere, which is difficult to be directly utilized by plants. PGPR can convert atmospheric N 2 to NH4 + by nitrogenase complex, for plant growth (Hoffman et al. 2014). Three nitrogen fixation-related genes (iscU, glnB, and glnA) were identified in the genome of the Rs-2 strain ( Table 2). The dimeric protein iscU plays a role in assembling iron-sulfur clusters in the nitrogen fixation pathway, and glnB, which encodes the P II protein genes, etc.
There were 24 genes related to siderophores biosynthesis and transmembrane carrier transport in the Rs-2 genome ( Table 3). The biosynthesis mainly was controlled by the operon iucABCD, and iucA and iucC are involved in the production of various siderophores such as achromobactin and deferoxamine. In addition, 20 siderophore transporter genes were found in the Rs-2 genome viz. fepA, fcuA, fhuA, and pfeA, etc.
The EcSMS35 1263 gene, which can encode D-cysteine desulfhydrase, was found in the Rs-2 genome ( Table 5). The BLAST alignment showed that the amino acid sequence of D-cysteine desulfhydrase was highly consistent with the sequence of ACCD.
Analysis showed that 19 genes related to organic acid synthesis, phosphatase, and inorganic phosphorus transport systems were found in the Rs-2 genome ( Table 6). The genes for organic acid synthesis mainly include the gcd gene encoding GDH and the PQQ synthesis gene pqqBCDEF, etc. Class B acid phosphatase encoding gene aphA and an alkaline phosphatase-related gene and a triphosphatase-encoding gene, etc.

Pot trials
The results of pot experiments further verified the growthpromoting properties of Rs-2 (Fig. 6). When radish was grown in pots with Rs-2, the fresh and dry weights of shoots and roots, root length, and the number of leaves of radish plants increased significantly (Fig. 7). The fresh and glnA Glutamine synthetase Table 3 Annotation of Siderophore production-related genes in Rs-2 Plant growth promoting characteristics ID Gene Function annotation Siderophore production and transportation GE01888 iucA Aerobactin siderophore biosynthesis protein IucA GE01889 iucB Hydroxylysine acetyltransferase GE01890 iucC Aerobactin synthase IucC GE01891 iucD L-lysine N 6 -monooxygenase GE00386 fepA TonB-dependent siderophore receptor GE00329 fcuA TonB-dependent receptor GE00060 fhuA TonB-dependent siderophore receptor GE00599 pfeA TonB-dependent siderophore receptor GE01483 yncD TonB-dependent siderophore receptor GE01610 tonB Outer membrane transport energization protein TonB GE00777 yvrB ABC-type Fe 3+ -siderophore transport system, permease component GE00392 fepD ABC-type Fe 3+ -siderophore transport system, permease component GE00776 fecE ABC-type cobalamin/Fe 3+ -siderophores transport systems, ATPase components GE00398 entA 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase GE00396 entE 2,3-dihydroxybenzoate-AMP ligase GE01608 pfeA Outer membrane receptor protein GE01253 fhuE Ferric-rhodotorulic acid transporter GE04111 fhuF Ferric iron reductase involved in ferric hydroximate transport GE04064 foxA Ferrioxamine B receptor GE02837 yusV ABC transporter GE00063 fhuB Iron-hydroxamate transporter permease subunit GE00347 Fct Hypothetical protein GE02882 yqjH Siderophore-interacting protein GE01892 iutA Ligand-gated channel protein  (Fig. 7a); the fresh and dry weights of roots increased by 78.23 and 67.35%, respectively (Fig. 7b); and the number of leaves increased significantly. The leaf area was larger compared to that of CK; the root length was 15.18 ± 2.52 cm (Fig. 7c). Selvaraj et al. (2008) found that treatment of seeds with phosphate-dissolving strains increased root elongation and biomass. It is indicated that the application of Rs-2 may promote the growth of radishes by promoting the elongation of the root system of radishes to better cope with the nutrient-deficient environment.
In addition, the plants in pots with Rs-2 have more vivid leaves, and the chlorophyll in leaves was extracted. The results of one-way ANOVA showed that there were significant differences in the contents of chlorophyll and carotenoid C among different treatments (p < 0.05, n = 6). The average values of chlorophyll A, chlorophyll B, total chlorophyll and carotenoid C in radish plants treated with Rs-2 were 0.69 ± 0.08 mg/g, 0.30 ± 0.03 mg/g, 0.99 ± 0.15 mg/g, and 0.48 ± 0.05 mg/g, respectively (Fig. 8a), which will be more beneficial to the photosynthesis of plants. Liu et al. (2015) demonstrated that phosphorus-solubilizing bacteria 9320-SD could effectively increase the chlorophyll content of maize after the phosphorus-solubilizing ability was enhanced, which may be related to the fact that PGPR promotes the uptake and assimilation of nutrients such as P by plants. In addition, the total phosphorus in the shoots and roots of radish plants were measured, respectively. The total phosphorus concentration in the shoots and roots of the plants with the effect of Rs-2 was increased by 30.00% (p = 0.001, n = 6) and 37.52% (p = 0.000001, n = 6), respectively, compared with the CK treatment, and the average values were 9.07 ± 0.61 mg/g and 6.61 ± 0.35 mg/g, respectively (Fig. 8b). Vessey and Heisinger (2001) found that Penicillium bilaii increased pea root length by 48%, root dry weight by 13%, and phosphorus concentration in the upper part of the root by 13% in the treatment without phosphorus fertilization. This effect is only significant under P-limited conditions. Furthermore, a higher concentration of available phosphorus was detected in the potting soil treated with Rs-2, at a concentration of 0.05 ± 0.01 mg/g, which was 1.75 times that of the control group (Fig. 8c). These results indicate that Rs-2 has excellent phosphorus-dissolving ability in phosphorus-deficient environments and promotes phosphorus uptake by radish plants to meet their nutrient requirements.

Discussion
The results of the genomic analysis showed that Rs-2 has general characteristics of Enterobacter species (Panigrahi et al. 2020;Naushad et al. 2019); COG has determined the  pstB Phosphate import ATP-binding protein PstB transport and metabolism of carbohydrates and amino acids, transcription and inorganic salt transport, etc., which showed that Rs-2 can generate a variety of secondary metabolites, and can regulate the transport of inorganic salt ions to help plants resist salt stress.
Enterobacter cloacae are reported beneficial to plant growth, but some unfriendly human opportunistic pathogens or phytopathogenic bacteria were also present. The virulence of these pathogens relies on plasmid-encoded type III secretion systems (T3SS) or type IV secretion systems (T4SS), which lead to the disruption of host defense mechanisms by secreting and transferring effector proteins (Christie and Vogel 2000;Ren et al. 2010). There were no virulence factors related to T3SS and T4SS were found in the genome of Rs-2. In addition, Rs-2 showed significant growth-promoting advantages in cotton applications, especially in reducing ethylene production in cotton plants (Wu et al. 2012). In coculture with Bacillus subtilis SL-44, it showed an inhibitory effect on the growth of Rhizoctonia solani. Although the type VI secretion system (T6SS) gene was significantly upregulated, it may be mainly manifested in the competition for regulatory bacteria (Li et al. 2021), such as injecting toxins into the cells of competing bacteria and protecting autoimmune proteins. In the study of the interaction between Rs-2 and plants, no pathogenicity to plants was found.
The drug susceptibility test results are basically consistent with the susceptibility of Enterobacter cloacae to quinolones reported in other studies (Liu et al. 2021), but there are differences in the susceptibility to cephalosporins and a few other drugs, which may be due to the individual difference. The drug resistance of Enterobacter cloacae, which has been domesticated for a long time in the laboratory, may also change with genetic changes. Resistance to drugs such as clindamycin and furazolidone (lentine) provides favorable conditions for Rs-2 to compete with other coexisting bacteria or fungi in the soil rhizosphere.
Iron-sulfur clusters were contained in all cellular organisms. They were indispensable in the basic metabolic pathways of photosynthesis, respiration, and nitrogen fixation. The dimeric protein iscU was found in the Rs-2 genome, which can act as a scaffold for the assembly of iron-sulfur clusters and deliver to target proteins (Agar et al. 2000), such as ferritin and iron-molybdenum proteins during biological nitrogen fixation. The P II protein encoded by the glnB gene is necessary for nitrogen fixation. Li et al. (2001) found that Azospirillum brasilense lost its nitrogen fixation ability when the glnB gene is mutated. In addition, the P II protein may participate in nitrogen fixation regulation by regulating the activity of NifA (de Zamaroczy 1998). However, in Klebsiella pneumoniae, the P II protein also regulates nitrogen fixation, while it may be not necessary since the glnB gene mutation has no effect on nitrogenase activity (Merrick and Edwards 1995). Siderophore is a chelating factor secreted by bacteria to meet the needs of their own life activities in a low iron environment, and has a strong binding specificity to Fe 3+ . Siderophore biosynthesis requires the activities of the acetyltransferase iucD and the hydroxylase iucB to form N 6 -acetyl-N 6 -hydroxylysine (ahLys) (Mydy et al. 2020). When bacteria exist in an iron-deficient environment, fepA transports iron to the cell membrane by adsorbing the ferric enterobactin complex on the cell surface (Wang and Liu 2009). Iron trapping systems exist in some bacteria, and these systems typically include enzymes that synthesize and secrete siderophore-enterobactin and the ferric enterobactin receptor protein fepA. In iron-deficient MSA medium, Rs-2 produced higher levels of siderophore than in iron-rich environments (Fig. 5c), suggesting that iucB, iucD, fepA, etc. play important roles in siderophore synthesis and transport. Many studies have shown that IAA secreted by PGPR can regulate plant growth even at low levels, specifically in promoting cell elongation and division, inducing plant disease resistance, etc. (Mano and Nemoto 2012). PGPR synthesizes IAA through various pathways, viz. indoleacetamide pathway, indolepyruvic acid pathway, tryptamine pathway, indoleacetonitrile pathway, tryptophan side chain pathway, etc., which depend on tryptophan (Liu et al. 2021). The ipdC gene was found by searching the IAA biosynthetic pathway in the Rs-2 genome ( Table 4). The ipdC gene is crucial in the synthesis of indole pyruvate in the indole pyruvate pathway. In the indole-3-pyruvate (IpyA) pathway, tryptophan is oxidatively deaminated to indolepyruvate, which is then oxidized to indoleacetaldehyde by the action of indolepyruvate decarboxylase (encoded by ipdC), which is then oxidized to IAA (Imada et al. 2017). Costacurta et al. (1994) found that the ipdC gene can regulate the production of IAA, and when the ipdC gene of Sp245 was interrupted, the secretion of IAA was reduced by 90% compared with the wild-type strain. In addition, the aux2 gene encoding indoleacetamide hydrolase in the indoleacetamide pathway, the iaaH gene involved in encoding indole-3-acetyl-aspartic acid hydrolase, and the tryptophan biosynthesis protein trpC were also retrieved (Table 4).
PGPR utilizes self-synthesized ACCD to degrade ACC, the direct precursor of ethylene synthesis, into ammonia and α-Ketobutyric acid (Glick 2014), reducing the amount of ethylene synthesis, thus avoiding the inhibitory effect of high concentration of ethylene on root elongation and increase of stress resistance of plants (Bhattacharyya and Jha 2012). Riemenschneider et al. (2005) reported that D-cysteine desulfhydrase had the ability to convert D-cysteine to pyruvate, H 2 S, and ammonia without ACCD activity. But McDonnell et al. (2009) subsequently showed that D-cysteine desulfhydrase indeed had ACCD activity because it is a dual-function enzyme. No other genes related to ACCD synthesis were found in the Rs-2 genome except EcSMS35 1263, but Rs-2 was confirmed to have the ability to synthesize ACCD, so we think that D-cysteine desulfurase may have ACCD activity.
Gluconic acid secreted by phosphate-solubilizing microorganisms (PSMs) is considered to be one of the main mechanisms for dissolving IP. The secretion of gluconic acid is mainly derived from the glucose oxidation of PSMs, which requires the catalysis of GDH, and GDH usually requires the oxidation cofactor PQQ to catalyze the production of gluconic acid. Many kinds of literature have shown that GDH was an enzyme encoded by the gcd gene (Rasul et al. 2019), which exists in Rs-2 and plays an important role in the secretion of gluconic acid. PQQ is encoded by the pqq operon and can catalyze the production of gluconic acid from glucose after binding to quinone protein glucose dehydrogenase. Mutations in the gcd or pqq genes result in the inability of PSMs to secrete gluconic acid (Boiardi et al. 1996). This is consistent with our findings that the Rs-2 genome contains a complete gluconic acid secretion system, which shows a good ability to dissolve phosphorus in PVK medium and in phosphorus-deficient soil.
Organic phosphorus is as difficult as inorganic phosphorus to be absorbed by plants. Organic phosphorus is mineralized by enzymatic hydrolyses, such as phosphatase, and releases inorganic phosphoric acid by hydrolyzing phospholipid bonds. Non-specific phosphatases were classified into three categories: A, B, and C, according to the phosphatase genes and characteristics. Acid phosphatase (aphA), found in Rs-2, is a nonspecific class B acid phosphatase that removes the phosphate group of organophosphate monoesters to generate phosphate ions and hydroxyl groups. Three phosphatase-encoding genes in Rs-2 indicate that it has great potential in the hydrolysis of organophosphorus, which is of great research value.
Previous studies reported that there are two main inorganic phosphate transport systems in bacteria, the phosphate inorganic transport (Pit) system and the phosphate specific transfer (Pst) system (Rosenberg 1987), which are responsible for the absorption and transport of inorganic phosphate in the soil. Pit systems can be powered by proton-motive force to support the transport of HPO 4 2− in a high inorganic phosphate environment (Santos-Beneit et al. 2008). The Pst system of high-affinity inorganic phosphate is relatively complex and generally composed of PstC, PstS, PstB, PstA, and PhoU proteins (Amemura et al. 1985;Sorger-Herrmann et al. 2015), which were detected in the Rs-2 genome. Studies have shown that bacteria bind inorganic phosphate in the environment through the PstS protein and use the integral membrane proteins PstC and PstA to form a transmembrane channel to transport inorganic phosphate across the membrane. During this process, ATP is hydrolyzed by the ATPbinding protein PstB, and the released energy is used as the transport power (Chan and Torriani 1996). In the Pst system, phoU protein plays a negative regulatory role in inorganic phosphate signaling (Rice et al. 2009), and phosphorylated phoB can also regulate the expression of the Pho regulon gene (Lamarche et al. 2008).
We screened 51 genes related to bacteria-plant interactions in the whole genome of Rs-2. Through gene function analysis, we determined that these genes were mainly related to nitrogen fixation, siderophore production, IAA secretion, ACCD activity, and phosphorus-solubilizing growth-promoting properties. And the plant growth-promoting properties were confirmed by in vitro experiments of Rs-2.
Pot experiments further demonstrated the great growthpromoting potential of Rs-2, which is mainly manifested in the ability to dissolve phosphate, although this may involve the production of phytohormones or other mechanisms (nitrogen fixation, siderophore production, IAA secretion, etc.). Radishes can grow normally and are not pathogenic during the symbiotic process with Rs-2, which indicates that Rs-2 is different from phytopathogenic bacteria such as Enterobacter cloacae EcWSU1 (Humann et al. 2011) and is a beneficial microorganism.

Conclusions
This study was conducted to demonstrate the plant growthpromoting characteristics of E. cloacae Rs-2, correlating genotypic and phenotypic characteristics. The results of whole-genome sequencing showed that the genes related to plant growth-promoting, such as nitrogen fixation, siderophore production, IAA and ACCD secretion, and phosphorus dissolution. The results of the qualitative and quantitative analysis showed that Rs-2 has considerable siderophore capacity, and the secretion concentration of IAA and ACCD was also high. In addition, Rs-2 secreted a variety of lowmolecular-weight organic acids, which have good solubility for IP. In pot experiments, Rs-2 as a soil inoculant improved the resistance of radish plants to abiotic stress environments. The genetic screening and analysis provided by this study will contribute to further understanding and improvement of E. cloacae, establishing the prospect of increasing the yields of the crop in validation field trials.