Genetic Organization of the aprX-lipA2 Operon Affects the Proteolytic Potential of Pseudomonas Species in Milk

Abstract

Psychrotolerant <i>Pseudomonas</i> species are a main cause of proteolytic spoilage of ultra-high temperature (UHT) milk products due to the secretion of the heat-resistant metallopeptidase AprX, which is encoded by the first gene of the <i>aprX-lipA2</i> operon. While the proteolytic property has been characterized for many different <i>Pseudomonas</i> isolates, the underlying <i>aprX-lipA2</i> gene organization was only described for a few strains so far. In this study, the phylogenomic analysis of 185 <i>Pseudomonas</i> type strains revealed that the presence of <i>aprX</i> is strongly associated to a monophylum composed of 81 species, of which 83% carried the <i>aprX</i> locus. Furthermore, almost all type strains of known milk-relevant species were shown to be members of the three monophyletic groups <i>P. fluorescens</i>, <i>P. gessardii</i>, and <i>P. fragi</i>. In total, 22 different types of <i>aprX-lipA2</i> genetic organizations were identified in the genus, whereby 31% of the species tested carried the type 1 operon structure consisting of eight genes (<i>aprXIDEF prtAB lipA2</i>). Other genetic structures differed from type 1 mainly in the presence and location of genes coding for two lipases (<i>lipA1</i> and <i>lipA2</i>) and putative autotransporters (<i>prtA</i> and <i>prtB</i>). The peptidase activity of 129 strains, as determined on skim milk agar and in UHT-milk, correlated largely with different <i>aprX-lipA2</i> gene compositions. Particularly, isolates harboring the type 1 operon were highly proteolytic, while strains with other operon types, especially ones lacking <i>prtA</i> and <i>prtB</i>, exhibited significantly lower peptidase activities. In conclusion, the phylogenomic position and the <i>aprX-lipA2</i> gene organization specify the proteolytic potential of <i>Pseudomonas</i> isolates. In addition, however, an interplay of several environmental factors and intrinsic traits influences production and activity of AprX, leading to strain-specific proteolytic phenotypes.