The abundant nutrients (e.g. lipids, proteins, amino acids, iron and other micronutrients) as well as high water activity in meat provide suitable conditions for the growth of spoilage bacteria (Doulgeraki et al., 2012). Within the meat ecosystem, the bacteria that ultimately dominate are described as specific spoilage organisms (SSOs), which mediate spoilage through their metabolic activities (Nychas et al., 2008) and cause substantial economic losses and food waste (Odeyemi et al., 2020).

Pseudomonas spp. are more likely to dominate chilled meat products under aerobic conditions due to their psychrotrophic nature and prolific production of lipases and proteases (Mansur et al., 2019; Wickramasinghe et al., 2019; Yang et al., 2022; Yang et al., 2023). Moreover, their efficient glucose utilization, siderophore production, and low oxygen affinity confer a competitive advantage in nutrient acquisition (Gram et al., 2002; Mohareb et al., 2015; Tsigarida et al., 2003). The genus Pseudomonas exhibits characteristic intraspecific diversity, encompassing several prominent spoilage species during meat refrigeration, such as Pseudomonas fragi, Pseudomonas putida, Pseudomonas lundensis, and Pseudomonas fluorescens (Lalucat et al., 2022; Longhi et al., 2022; Martins et al., 2006; Wickramasinghe et al., 2019). While their metabolic activities and role in spoilage are well-documented, previous studies have largely overlooked the potential intraspecific interactions between Pseudomonas species that contribute to co-spoilage phenomena.

Microbial interactions—such as antagonism, mutualism, and commensalism—are known to influence the characteristics of meat spoilage (Gram et al., 2002; Nychas et al., 2008). For example, the interactions between Pseudomonas and other genera, such as Brochothrix thermosphacta (Fang et al., 2022; Zhou et al., 2024), Photobacterium (Hauschild et al., 2022), and Acinetobacter (Wang and Xie, 2020), have been increasingly recognized as playing significant roles in meat spoilage. Identifying the key metabolic genes responsible for spoilage compound production is essential for developing mitigation strategies (Mohareb et al., 2015). Transcriptomic studies indicate that microbial interactions can significantly alter gene expression. For instance, quorum sensing (QS) in Hafnia alvei was shown to modulate gene expression and biofilm formation when co-cultured with P. fluorescens (Wang et al., 2024a), while co-inoculation of Pseudomonas weihenstephanensis and Macrococcus caseolyticus enhanced proteolytic capacity through differential regulation of apr-related genes (Zhang et al., 2024b). Moreover, targeted gene knockout approaches have enabled functional validation of spoilage-related genes, including those involved in siderophore biosynthesis, QS regulation, and extracellular protease secretion in Pseudomonas spp. (Wang et al., 2021; Cui et al., 2023; Wang et al., 2024b).

Despite these advances, Pseudomonas species often coexist in meat ecosystems, yet the molecular mechanisms governing their interspecific interactions and the specific genetic determinants driving cooperative spoilage remain poorly understood. Core genes within the Pseudomonas spp., including those involved in ribosome synthesis and basal metabolism, are generally conserved, while interspecific variation in their expression exists and genetic divergence is further driven by horizontal gene transfer and niche adaptation (Koehorst et al., 2016). This metabolic diversity, along with shared pathways among Pseudomonas species, may underlie the complex microbial interactions that enhance their co-spoilage potential (Zhuang et al., 2021).

Our previous work investigated the co-spoilage potential of different Pseudomonas combinations in chilled pork and found that the combination of P. fragi + P. putida exhibited a synergistic effect that accelerated meat spoilage (Shang et al., 2025). However, the molecular basis of this synergy remains largely unknown. Therefore, this study employed an integrated approach combining comparative transcriptomics and targeted gene knockout to elucidate the molecular mechanisms underlying the synergistic co-spoilage interaction between P. fragi and P. putida, and to identify key genes contributing to enhanced spoilage potential in chilled meat.