Abstract
Milk and dairy products harbor a wide variety of bacterial species that compete for both limited resources and space. Under these competitive conditions, bacteria develop specialized mechanisms to protect themselves during niche colonization and nutrient acquisition processes. The bacterial antagonism mechanisms include the production of antimicrobial agents or molecules that facilitate competitor dispersal. In the present work, a bacterial strain designated RC6 was isolated from Ricotta and identified as Bacillus cereus. It generates antimicrobial peptide (AMP) when grown in the presence of casein. The AMP was active against several species of Bacillus and Listeria monocytogenes. MALDI-TOF analysis of the RP-HPLC purified fractions and amino acid sequencing revealed a molecular mass of 751 Da comprised of a 6-residue sequence, YPVEPF. BLAST analysis showed that the AMP corresponds to the fractions 114–119 of bovine β-casein and represents the product of a specific proteolysis. Analysis of the purified proteolytic fractions from the B. cereus RC6 culture supernatant indicated that the presence of at least two different endoproteases is crucial for the generation of the AMP. Indeed, we were able to identify two new candidate endoproteases by means of genome sequencing and functional assignment using a 3D structural model and molecular docking of misannotated hypothetical proteins. In this light, the capacity of B. cereus RC6 to generate antimicrobial peptides from casein, through the production of extracellular enzymes, presents a new model of antagonistic competition leading to niche colonization. Hence, as a dairy product contaminant, this strategy may enable proteolytic B. cereus RC6 niche specialization in milk matrices.
Highlights
Most natural environments harbor a wide variety of microbial species (Hibbing et al, 2010)
The highest antimicrobial activity was exhibited against closely related bacteria, including B. cereus ATCC 11778 (BC45) and B. thuringiensis USDA HD22
Observation of the B. cereus RC6 strain phenotype on skim milk/casein agar medium revealed a clear halo around the colony, indicating protease activity and raising the question of the possible involvement of this activity in casein degradation and antimicrobial peptide (AMP) generation
Summary
Most natural environments harbor a wide variety of microbial species (Hibbing et al, 2010). Bacteria compete with their neighbors for limited resources and niche space (Keymer et al, 2006; Ghoul and Mitri, 2016). The outcome of this interaction is the evolution of a diverse and powerful arsenal of biological arms. It is generally accepted that lactic acid bacteria (LAB), such as Lactobacillus and Leuconostoc, are the dominant fermentative population in milk and dairy products (Quigley et al, 2013). Psychotrophic microorganisms, yeast and coliform bacteria ( they are not part of the natural microbial population), are found in milk and cheese as spoilage microbes (SamarŽija and Pogacic, 2012). B. cereus has been isolated from raw milk, pasteurized milk, and cheddar cheese (Ahmed et al, 1983), while dried milk products are known to be frequently contaminated with B. cereus (Becker et al, 1994)
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