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The extracellular matrix is essential for the biofilm formation of food spoilers. Pseudomonas fluorescens PF07 is a previous isolate from spoiled marine fish; however, the genes involved in the extracellular matrix formation of PF07 biofilms remain poorly defined. In this study, PF07 formed a wrinkled macrocolony biofilm through the high production of extracellular matrix. The genes involved in biofilm matrix formation and regulation were screened and identified by RNA-seq-dependent transcriptomic analysis and gene knock-out analysis. The macrocolony biofilms of PF07 grown for 5 days (PF07_5d) were compared with those grown for 1 day (PF07_1d). A total of 1,403 genes were significantly differentially expressed during biofilm formation. These mainly include the genes related to biofilm matrix proteins, polysaccharides, rhamnolipids, secretion system, biofilm regulation, and metabolism. Among them, functional amyloid genes fapABCDE were highly upregulated in the mature biofilm, and the operon fapA-E had a –24/–12 promoter dependent on the sigma factor RpoN. Moreover, the RNA-seq analyses of the rpoN mutant, compared with PF07, revealed 159 genes were differentially expressed in the macrocolony biofilms, and fapA-E genes were positively regulated by RpoN. In addition, the deletion mutants of fapC , rpoN , and brfA (a novel gene coding for an RpoN-dependent transcriptional regulator) were defective in forming mature macrocolony biofilms, solid surface-associated (SSA) biofilms, and pellicles, and they showed significantly reduced biofilm matrices. The fap genes were significantly downregulated in Δ brfA , as in Δ rpoN. These findings suggest that the functional amyloid Fap is the main component of PF07 biofilm matrices, and RpoN may directly regulate the transcription of fap genes, in conjunction with BrfA. These genes may serve as potential molecular targets for screening new anti-biofilm agents or for biofilm detection in food environments. [ABSTRACT FROM AUTHOR]

The extracellular matrix is essential for the biofilm formation of food spoilers. Pseudomonas fluorescens PF07 is a previous isolate from spoiled marine fish; however, the genes involved in the extracellular matrix formation of PF07 biofilms remain poorly defined. In this study, PF07 formed a wrinkled macrocolony biofilm through the high production of extracellular matrix. The genes involved in biofilm matrix formation and regulation were screened and identified by RNA-seq-dependent transcriptomic analysis and gene knock-out analysis. The macrocolony biofilms of PF07 grown for 5 days (PF07_5d) were compared with those grown for 1 day (PF07_1d). A total of 1,403 genes were significantly differentially expressed during biofilm formation. These mainly include the genes related to biofilm matrix proteins, polysaccharides, rhamnolipids, secretion system, biofilm regulation, and metabolism. Among them, functional amyloid genes fapABCDE were highly upregulated in the mature biofilm, and the operon fapA-E had a –24/–12 promoter dependent on the sigma factor RpoN. Moreover, the RNA-seq analyses of the rpoN mutant, compared with PF07, revealed 159 genes were differentially expressed in the macrocolony biofilms, and fapA-E genes were positively regulated by RpoN. In addition, the deletion mutants of fapC, rpoN, and brfA (a novel gene coding for an RpoN-dependent transcriptional regulator) were defective in forming mature macrocolony biofilms, solid surface-associated (SSA) biofilms, and pellicles, and they showed significantly reduced biofilm matrices. The fap genes were significantly downregulated in ΔbrfA, as in ΔrpoN. These findings suggest that the functional amyloid Fap is the main component of PF07 biofilm matrices, and RpoN may directly regulate the transcription of fap genes, in conjunction with BrfA. These genes may serve as potential molecular targets for screening new anti-biofilm agents or for biofilm detection in food environments.

Frozen chicken nuggets can be stored over a long period, during which time they may be exposed to temperature abuse conditions leading to spoilage by psychrophilic filamentous fungi. The purpose of this study was to investigate the main sources of chicken nugget spoilage. Mycological analyses were performed using raw materials and products from each stage of processing, as well as from the industry’s indoor air environment. The species were identified through observation of macroscopic and microscopic characteristics of isolates. The powder coating of samples showed counts between 10 1 and 10 4 CFU/g, predominantly of the species Penicillium polonicum , Aspergillus flavus , Aspergillus candidus , Aspergillus (Eurotium) amstelodami and Penicillium citrinum . During chicken nugget processing, a reduction in fungal counts to 10 1 CFU/g was observed, with P. polonicum as the most dominant species present. Penicillium glabrum was the only species isolated from the final product analyzed (10%). Additionally, it was also the predominant species in the factory’s air environment. According to the results, two possible sources of contamination of frozen chicken nuggets were detected, one from the lack of quality of the powder coatings used and another from the lack of clean air from the factory environment between the stages of baking and packaging. [ABSTRACT FROM AUTHOR]

Summary Domestication is an excellent model for studies of adaptation because it involves recent and strong selection on a few, identified traits [ 1–5 ]. Few studies have focused on the domestication of fungi, with notable exceptions [ 6–11 ], despite their importance to bioindustry [ 12 ] and to a general understanding of adaptation in eukaryotes [ 5 ]. Penicillium fungi are ubiquitous molds among which two distantly related species have been independently selected for cheese making— P. roqueforti for blue cheeses like Roquefort and P. camemberti for soft cheeses like Camembert. The selected traits include morphology, aromatic profile, lipolytic and proteolytic activities, and ability to grow at low temperatures, in a matrix containing bacterial and fungal competitors [ 13–15 ]. By comparing the genomes of ten Penicillium species, we show that adaptation to cheese was associated with multiple recent horizontal transfers of large genomic regions carrying crucial metabolic genes. We identified seven horizontally transferred regions (HTRs) spanning more than 10 kb each, flanked by specific transposable elements, and displaying nearly 100% identity between distant Penicillium species. Two HTRs carried genes with functions involved in the utilization of cheese nutrients or competition and were found nearly identical in multiple strains and species of cheese-associated Penicillium fungi, indicating recent selective sweeps; they were experimentally associated with faster growth and greater competitiveness on cheese and contained genes highly expressed in the early stage of cheese maturation. These findings have industrial and food safety implications and improve our understanding of the processes of adaptation to rapid environmental changes. [ABSTRACT FROM AUTHOR]