Abstract
Pseudomonas fluorescens is a typical spoiler of proteinaceous foods, and it is characterized by high spoilage activity. The sigma factor RpoN is a well-known regulator controlling nitrogen assimilation and virulence in many pathogens. However, its exact role in regulating the spoilage caused by P. fluorescens is unknown. Here, an in-frame deletion mutation of rpoN was constructed to investigate its global regulatory function through phenotypic and RNA-seq analysis. The results of phenotypic assays showed that the rpoN mutant was deficient in swimming motility, biofilm formation, and resistance to heat and nine antibiotics, while the mutant increased the resistance to H2O2. Moreover, the rpoN mutant markedly reduced extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized fish juice at 4°C; meanwhile, the juice with the rpoN mutant showed significantly higher sensory scores than that with the wild-type strain. To identify RpoN-controlled genes, RNA-seq-dependent transcriptomics analysis of the wild-type strain and the rpoN mutant was performed. A total of 1224 genes were significantly downregulated, and 474 genes were significantly upregulated by at least two folds at the RNA level in the rpoN mutant compared with the wild-type strain, revealing the involvement of RpoN in several cellular processes, mainly flagellar mobility, adhesion, polysaccharide metabolism, resistance, and amino acid transport and metabolism; this may contribute to the swimming motility, biofilm formation, stress and antibiotic resistance, and spoilage activities of P. fluorescens. Our results provide insights into the regulatory role of RpoN of P. fluorescens in food spoilage, which can be valuable to ensure food quality and safety.
Highlights
Pseudomonas spp. are Gram-negative, rod-shaped, ubiquitous microorganisms with simple nutritional needs (Remenant et al, 2015)
The core portion from bp 4 to 1473 of the open reading frame (ORF) was deleted, and the rpoN mutant was verified via polymerase chain reaction (PCR) (Supplementary Figure 1) and sequencing
The results of this study illustrated the involvement of RpoN in regulating the swimming motility, biofilm formation, resistance to stress conditions and antibiotics, and spoilage activity of P. fluorescens
Summary
Pseudomonas spp. are Gram-negative, rod-shaped, ubiquitous microorganisms with simple nutritional needs (Remenant et al, 2015). They largely contribute to the food spoilage process, and among the genus, Pseudomonas fluorescens is a typical spoiler of proteinaceous raw foods stored under aerobic refrigerated conditions, especially aerobically chill-stored seafood (Xie et al, 2018), meat (Doulgeraki and Nychas, 2013), and milk (Polêto et al, 2019). Motility is required for colonization and competition in food systems (Wang et al, 2018); biofilms can form on surfaces of various foods or food industry equipment and are often difficult to be removed by normal sanitation procedures (Galié et al, 2018); and the bacterial resistance to stress conditions during food preparation and processing and to antibiotics during agricultural production leads to persistent contamination in food systems (Verraes et al, 2013; Liu X. et al, 2018). Understanding how the bacterium regulates its spoilage ability is vital; the knowledge related to the regulatory mechanisms of spoilage is still limited
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