CRISPR-Cas9 mediated adiA knockout in Hafnia paralvei: Implications for agmatine production and acid stress survival in a fermented dairy matrix.

Agmatine, the product of the decarboxylation of arginine, catalysed by arginine decarboxylase (ADC), is a bioactive compound that functions as a neuromodulator and co-transmitter and has gained increasing attention in recent years due to its therapeutic potential, particularly for its neuroprotective properties. Members of the genus Hafnia are the main agmatine producers in dairy products. In this regard, Hafnia is considered a beneficial microorganism due to its ability to enhance cheese organoleptic properties and its emerging probiotic potential, making it relevant for functional food development, specially agmatine-enriched dairy products. This study aimed to identify the genetic basis for agmatine production in Hafnia paralvei and to assess its role in bacterial fitness. Genomic analysis of the strain H. paralvei IPLA15029 revealed the presence of two genes encoding putative ADC enzymes, adiA and speA, however, organized slightly different than those in other enterobacteria. In some bacteria, ADC exists in two forms: one involved in polyamine biosynthesis, encoded by the constitutive speA gene, and another involved in acid stress resistance, encoded by the adiA gene, which is inducible under acidic conditions. In vivo experiments under controlled pH conditions showed that agmatine accumulation occurs exclusively under acidic conditions, which also stabilize the compound by preventing its catabolism to putrescine. Gene expression analysis revealed that adiA was transcribed as a monocistronic unit, and that in these conditions, adiA is the gene responsible for agmatine production. This was confirmed by generating an adiA knockout mutant after the implementation of the CRISPR-Cas9 system, marking the first successful application of this technology in the genus Hafnia. Moreover, the adiA knockout demonstrated that the encoded arginine decarboxylase is essential for survival under severe acid stress.