AB medium (3 g/L K₂HPO₄, 0.3 g/L MgSO₄⋅7H₂O, 1 g/L NH₄Cl, 0.15 g/L KCl, 1 g/L NaH₂PO₄, 0.01 g/L CaCl₂, 0.0025 g/L FeSO₄⋅7H₂O, 5 g/L casein hydrolyzed amino acids) was prepared as previously described (Zimmer et al., 2014). The overnight culture of P. fluorescens PF08 (OD₅₉₅ = 1) was diluted 1:100 in prepared AB culture medium, followed by the addition of 5 g/L of different carbon sources (glucose, xylose, sucrose, fructose, lactose, or maltose). All samples were incubated at 28°C until an OD₅₉₅ of 1 was reached. After centrifugation at 8000 × g for 10 min, the cells were collected for the analysis of rhlI/R transcription level using qPCR. Samples incubated in LB broth at 28°C were the control.

An overnight culture of P. fluorescens PF08 (OD₅₉₅ = 1) was diluted 1:100 in fresh LB broth (pH 7.0 ± 0.2) containing different mass concentrations of NaCl (0.5, 1, 3, or 5%). All samples were incubated at 28°C until an OD₅₉₅ of 1 was reached. After centrifugation at 8000 × g for 10 min, the precipitates were collected for further analysis of rhlI/R transcription level using qPCR.

An overnight culture of P. fluorescens PF08 (OD₅₉₅ = 1) was diluted 1:100 in fresh LB broth. The pH was adjusted to 5, 6, 7, 8, or 9 by addition of HCl or NaOH. All samples were incubated at 28°C until OD₅₉₅ was 1. After centrifugation at 8,000 × g for 10 min, the cells were collected for further analysis of rhlI/R transcription level using qPCR.

An overnight culture of P. fluorescens PF08 (OD₅₉₅ = 1) was diluted 1:100 in fresh LB broth (pH 7.0 ± 0.2). The samples were incubated at 4, 15, 25, or 37°C until OD₅₉₅ was 1. After centrifugation at 8000 × g for 10 min, the cells were collected for further analysis of rhlI/R transcription level using qPCR. Samples incubated in LB broth at 25°C were the control.

The LuxI/R type system, which is mainly composed of AHLs LuxI and specific receptor protein LuxR, is one of the most typical QS regulatory systems. The system has been found in many Gram-negative bacteria, including Burkholderia, Escherichia coli, Hafnia alvei, and Pseudoalteromonas (Weiss et al., 2008; Choudhary et al., 2013; Yu et al., 2019; Zhu et al., 2019). In this system, sufficient LuxI-secreted AHLs can interact with LuxR to form a complex that combines with the promoters of the downstream target genes to regulate their expression. This mediates the production of spoilage-required factors, including a variety of proteases (Galloway et al., 2011). Presently, the LuxI/R type system RhlI/R was detected in an organized operon in the genome of P. fluorescens PF08. In this system, rhlI encodes the AHLs synthetase, and rhlR encodes the response regulator. The RhlI/R system is reported to work as part of a cascade QS regulatory system to regulate the production of pyocyanin and chitinase in conjunction with the LasI/R system, in which the expression of rhlI/R is also positively regulated by the LasI/R system (Defoirdt, 2018). Interestingly, in this study, the genes encoding LasI/R were not found in the genome of P. fluorescens PF08, suggesting that the QS system RhlI/R may act as an independent system to accomplish the regulation of related factors, which is uncommon. This is consistent with previous observations (Steindler et al., 2009), who found that the two QS systems are not hierarchically organized and are both important for the colonization of P. aeruginosa on the rice rhizosphere.

During evolution, some Gram-negative bacteria have lost one or more LuxI-type AHL synthetases and have retained only unpaired LuxR-type regulator proteins. These proteins, termed LuxR solos or Orphan proteins, are still able to sense AHLs and regulate bacterial behavior accordingly. Thus, they have important roles in bacterial eavesdropping and inter- and intraspecific signaling (Hudaiberdiev et al., 2015). Therefore, exploring the luxR solo genes in the genome is valuable to understand the QS properties of P. fluorescens PF08. Eleven genes encoding LuxR family regulatory proteins were identified in the genome of PF08 in this study (Table 2). The response regulators PprA and PprB were identified as a two-component regulatory system of P. aeruginosa with the function to regulate virulence factor production and cell motility. Furthermore, some QS regulatory genes in P. aeruginosa, such as the las system and rhl system, are also mediated by this system, and most PprB-activated genes are activated by C₄-HSL (Dong et al., 2005). In the P. fluorescens PF08 genome, only a separate gene, pprB, encoding the regulator PprB was identified. It was annotated as a luxR solo type regulator in the NR database. The presence of pprB explains the greater sensitivity of PF08 to C₄-HSL than to other AHLs, which we previously demonstrated (Li T. et al., 2019). The hybrid histidine kinase RcsC and the response regulator RcsB are homologous to environmentally responsive two-component regulators capable of promoting biofilm formation and the appearance of fimbriae (Stout and Gottesman, 1990; Nicastro et al., 2009). In this study, the rcsB gene was identified as a luxR solo type regulator in the genome of PF08, which is helpful to further understand the regulation process of biofilm formation in PF08. The sdiA gene predicted to encode LuxR homologs to regulate the ftsQAZ locus and virulence factors EspD in E. coli and Salmonella enterica has also been identified in PF08 (Kanamaru et al., 2000). In addition, some genes encoding putative LuxR solo type regulator, such as the environmental sensing gene agmR and the virulence regulator gene bvgA, were also annotated in the PF08 genome. These findings could genetically explain the ability of PF08 to enhance its virulence and spoilage factors through AHLs eavesdropping (Li T. et al., 2019).

Flagellar driven motility is an effective way for bacteria to escape immune system components and fungicides. The motility is mainly activated by the QS system under environmental stress (Díaz et al., 2011). Pili can mediate the adhesion of bacteria to different surfaces, which is essential to initiate biofilm formation. Thus, pili pose risks in medical facilities and the food industry. Our preliminary study observed the good motility and biofilm formation ability of P. fluorescens (Li et al., 2018). Presently, we explored the genes associated with biofilm formation and motility of P. fluorescens PF08. A total of 34 genes related to flagella were identified. Of these, 13 belonged to the flg gene cluster located in 3 flagellar operons. These genes regulate the synthesis of flagella by sensing the integrity of the flagellar structure (Chilcott and Hughes, 2000). Gene clusters including fliD/S/T/E/F/G/H and fliL/M/O/P/Q/R encode the flagellar assembly proteins. These clusters were detected in P. fluorescens PF08 (Figure 1B). The FliF protein forms the M-ring embedded in the cytoplasmic membrane of the basal part of the flagellar. The integral membrane proteins located in the MS-C loop (FlhA, FlhB, FliP, FliQ, FliR, and FliO) and soluble proteins (FliL, FliH, and FliJ) form the output device of flagella. FlhA and FlhB proteins comprise the platform for the attachment of FliL and FliH. The corresponding response of bacteria to external environmental changes is called chemotaxis. Chemotaxis is regulated by a two-component system involving the methyl-accepting chemotaxis protein (MCP). MCP can sense the changes of chemical signals in the external environment and adjusts the motility of bacterial flagella by regulating the two-component system (Kulasekara et al., 2013). The histidine kinase CheA and response regulator CheY that perform signal transduction are important components of the two-component regulatory system. The genes encoding CheA and CheY were detected in P. fluorescens PF08 in this study, indicating that the bacteria can adapt to environmental changes.

Concerning biofilm establishment, gene clusters involved in the biosynthesis of type IV pili were detected in the P. fluorescens PF08 genome in this study. The genes can promote bacterial adhesion and biofilm formation. Some of these genes are highly conserved in Pseudomonas sp. Two examples are pilG and pilJ, which are required for pili extension and twitching motility (Darzins, 1993; DeLange et al., 2007). Cyclic bis (3′–5′) diguanylic acid (c-di-GMP) is a second messenger molecule commonly found in bacteria. c-di-GMP improves the synthesis of cellulose, a major biofilm component, and therefore plays an important role in regulating biofilm formation (Tischler and Camilli, 2004). Genes encoding the key enzymes in the synthesis and metabolism of c-di-GMP were detected in this study. These enzymes included diguanylate cyclase (GGDEF domain), c-di-GMP synthetase, and phosphodiesterase (EAL domain). Their presence suggests the existence of the c-di-GMP regulatory pathway in P. fluorescens PF08. Alginate and certain extracellular proteins form the reticular skeleton that maintains the mechanical stability of biofilms. These components are crucial for the extracellular polymer (Laue et al., 2006). In the initial stage of extracellular polymer formation, a high content of alginate is important in the attachment of bacteria to the surface. The presence of a series of alginate synthesis genes, including alg8, alg44, algA, algK, algE, algQ, and algX, and the aforementioned genes explains the strong biofilm formation and motility ability of P. fluorescens at the genetic level, which was confirmed in our previous study (Li et al., 2018).

Extracellular proteases secreted by spoilage bacteria can decompose proteins in food matrix into small nitrogen-containing substances to maintain their own growth, accompanied by rapid deterioration of food quality including taste and flavor. Protease is the most important spoilage factor. Therefore, knowledge of protease-related genes is necessary to better understand and inhibit food spoilage caused by P. fluorescens PF08. Presently, a total of 13 protease-encoding genes, including the metalloprotease aprX, alkaline protease aprA, ATP-dependent Clp protease clpP, Zn-dependent metalloprotease sprT, aspartyl protease asP1, and serine protease serP, were identified in the PF08 genome. Among these, alkaline proteases are the most important type of proteases that cause food spoilage. Scatamburlo et al. (2015) demonstrated that the alkaline proteases from Pseudomonas sp. cause the spoilage of goat milk. Similarly, bacterial metalloproteases have been implicated in the spoilage of diverse foods. Reducing the activity of metalloprotease AprX can improve the quality of ultra-high temperature-treated milk during storage (Zhang et al., 2019). Furthermore, the serine protease SerP in H. alvei H4 may be crucial in the spoilage of instant sea cucumber (Zhu et al., 2020). In addition to these typical proteases, other genes encode some spoilage-related lipases in PF08, such as lipase D (lipD), phospholipase (rssA), lysophospholipase (pldB), and triacylglycerol esterase (estA). These lipases can degrade fats and lipoproteins of aquatic products to produce free fatty acids, glycerol, and choline, thus accelerating spoilage (Glantz et al., 2020). Choline can be further converted into harmful end products like dimethylamine and trimethylamine, which detrimentally affect the flavor of aquatic products. The foregoing genes found in the P. fluorescens PF08 genome confirmed the spoilage potential of the bacterium from a genetic perspective.

Some metabolic pathways in bacteria are responsible for further metabolizing proteins, carbohydrates, and lipids. Various substances produced by their degradation form volatile components, such as trimethylamine, putrescine, and hydrogen sulfide. These volatiles are associated with putrid off-odors in aquatic products and are markers of spoilage. Pathways of amino acid metabolism, sulfur metabolism, and amine metabolism are therefore considered to be involved in food spoilage. The genetic information related to these relevant metabolic pathways was presently analyzed to explain the spoilage potential of P. fluorescens PF08. As shown in Figure 3C, amino acid metabolism was the primary function of P. fluorescens PF08 in KEGG annotations, suggesting good potential for protein degradation. The Cyc gene cluster is an important gene cluster involved in sulfur metabolism. Genes in the cluster encode key enzymes, including adenylylsulfate kinase CysC and phosphoadenosine phosphosulfate reductase CysH. These enzymes catalyze the multi-step reduction of adenosine-5’-phosphosulfate to hydrogen sulfide in PF08. Other genes associated with sulfur metabolism that were detected in the PF08 genome included glpE (encoding thiosulfate sulfurtransferase), ssuD (encoding alkanesulfonate monooxygenase), and sseA (encoding 3-mercaptopyruvate sulfurtransferase). Their presence is indicative of sulfur metabolic activity of PF08.

Amine metabolism of SSOs is another important metabolic pathway involved in the spoilage of aquatic products. This pathway was also analyzed in this study. Putrescine is one of the main causes of the unpleasant smell of rotten aquatic products. The genes encoding a series of proteins responsible for the production of putrescine, such as putrescine transport system permease protein, putrescine-binding periplasmic protein E, putrescine aminotransferase, and gamma-glutamylputrescine oxidase, were identified in the ammonia metabolism pathway of P. fluorescens PF08. Genes encoding trimethylamine dehydrogenase and spermidine synthase were also detected. These enzymes are involved in trimethylamine metabolism and spermidine production, respectively. The results demonstrate that PF08 has the potential for amine metabolism. Interestingly, although Gloria et al. (2011) detected cadaverine and histamine in spoilage milk inoculated with P. fluorescens; however, no genes related to cadaverine and histamine production were found in P. fluorescens PF08 in this study.

The continuous consumption of proteins and ions caused by microbial metabolism leads to the irreversible deterioration of the food microenvironment. To adapt to this change of the environment, microorganisms adjust their metabolic activities to maintain their normal growth by secreting some proteins involved in homeostasis. We have demonstrated that P. fluorescens PF08 is the SSO of turbot during low temperature storage, suggesting that this bacterium has a particular advantage in interbacterial competition during this process (Li T. et al., 2019). Investigation of genes related to the stress response in the P. fluorescens PF08 genome is helpful to reveal the reasons behind this advantage. RpoS and RelA were detected in the genome. They are considered typical environmental response regulators involved in stress survival in bacteria. Liu et al. (2018) found the RpoS-deficient mutant strains of P. fluorescens were less tolerant to environmental factors of high temperature, low pH, and starvation. The QS system of the mutant strains was also suppressed. In P. fluorescens PF08, the rpoS gene may play a similar role. Pyoverdine is a low molecular weight organic compound that can bind Fe²⁺ efficiently under low iron stress conditions to maintain the necessary Fe²⁺ uptake in cells. The synthesis of pyoverdine in bacteria is also regulated by the QS system (Xu et al., 2019). The pvdA and pvdE genes regulate the biosynthesis of pyoverdine. The presence of both genes in PF08 indicate that PF08 may have a certain growth advantage in low iron stress. Other genes identified in PF08 included some general stress response-related genes, including the antioxidant protein-coding gene ahpC, glutathione peroxidase-coding gene bsaA, starvation stringent protein-coding genes sspA and sspB, and heavy metal detoxification-related genes cusS and merR. The existence of these genes may be a potential reason for the environmental adaptability and competitive advantage of PF08.

The accumulation of antibiotics in the environment and animal food farming processes imposes antibiotic pressure on bacteria, which had driven the evolution of a series of resistance pathways. Bacterial drug resistance can be transmitted by conjugation transfer, which poses a great challenge to food safety. More than 40 drug resistance-related genes were found in the genome of PF08. Hydrolysis of antibiotics into harmless metabolites by secretion of some key enzymes is the main pathway of bacterial drug resistance. β-lactamase and penicillin amidase encoded in PF08 can accelerate the metabolism of intracellular penicillin, thereby enhancing the tolerance of PF08 to penicillin. The gene encoding D-amino-acid oxidase (DAO), that can degrade cephalosporin, was also detected in PF08. In addition, the rapid efflux through the transport system of antibiotics that are not degraded is another effective means of bacterial survival. A total of 12 ABC-type multidrug transport system-related genes and 24 multidrug efflux pump genes were identified in the PF08 genome. The system composed of these coding genes can efficiently recognize specific lipophilic antibiotics and transport them out of the cells. The discovery of these genes has contributed to a better understanding of the drug resistance of PF08.

The antibacterial properties of the metabolites of PF08 were also investigated. This antibacterial property was implied by the observed advantage of PF08 in competing with other bacteria for survival during turbot spoilage. As shown in Supplementary Figure 2, the ethyl acetate extract of PF08 produced significant zones of growth inhibition in plates inoculated with S. aureus and A. salmonicida. The findings indicated the bacteriostasis of metabolites by PF08. Indole alkaloids are one of the marine alkaloids. They are derived mainly from marine organisms, such as sponges and mosses, which have significant antibacterial, antitumor, and other biological activities. The genes involving in the biosynthesis of indole alkaloids were detected in PF08, indicating the potential of PF08 to synthesize alkaloids. Polyketides are natural compounds produced by bacteria and fungi. The various polyketides include macrolides, tetracyclines, and polyethers. These compounds have antibacterial and anti-infection activities. In bacteria, the biosynthesis of polyketides is regulated by polyketide synthase. Construction of the carbocyclic scaffold catalyzed by polyketide cyclase is a key step in the biosynthesis. The genes encoding polyketide synthase and polyketide cyclase were detected in PF08, as were antibiotic biosynthesis monooxygenase (ABM) superfamily genes. Qin et al. (2019) identified the non-catalytic role of ABM superfamily proteins in polyketide synthesis in maintaining the fidelity of the biosynthetic pathway. The results suggest that PF08 may have the ability to secrete this kind of antibacterial substance. These antimicrobial substances produced by PF08 are mostly lipophilic and can be transported extracellularly using their own drug efflux pump system to reduce intracellular accumulation and thus inhibit the growth of competing microorganisms without damaging themselves.