Abstract
Psychrotolerant Pseudomonas 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 aprX-lipA2 operon. While the proteolytic property has been characterized for many different Pseudomonas isolates, the underlying aprX-lipA2 gene organization was only described for a few strains so far. In this study, the phylogenomic analysis of 185 Pseudomonas type strains revealed that the presence of aprX is strongly associated to a monophylum composed of 81 species, of which 83% carried the aprX locus. Furthermore, almost all type strains of known milk-relevant species were shown to be members of the three monophyletic groups P. fluorescens, P. gessardii, and P. fragi. In total, 22 different types of aprX-lipA2 genetic organizations were identified in the genus, whereby 31% of the species tested carried the type 1 operon structure consisting of eight genes (aprXIDEF prtAB lipA2). Other genetic structures differed from type 1 mainly in the presence and location of genes coding for two lipases (lipA1 and lipA2) and putative autotransporters (prtA and prtB). The peptidase activity of 129 strains, as determined on skim milk agar and in UHT-milk, correlated largely with different aprX-lipA2 gene compositions. Particularly, isolates harboring the type 1 operon were highly proteolytic, while strains with other operon types, especially ones lacking prtA and prtB, exhibited significantly lower peptidase activities. In conclusion, the phylogenomic position and the aprX-lipA2 gene organization specify the proteolytic potential of Pseudomonas 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.
Highlights
Psychrotolerant bacteria, predominant in cold stored raw milk, are known to secrete heat-resistant enzymes, which partly even withstand ultra-high temperature processing (von Neubeck et al, 2015; Glück et al, 2016)
As of March 2019 the List of Prokaryotic names with Standing in Nomenclature (LPSN; Parte, 2014) contained 188 Pseudomonas species with validly described names, of which P. brassicacearum, P. chlororaphis, P. oleovorans, and P. syringae can be further divided into nine subspecies (Supplementary Table S5)
A total of 185 type strains representing 87.8% (165) of the Pseudomonas species with validly published names were used and a maximumlikelihood (ML) phylogenomic tree was calculated on the basis of 92 universal bacterial core genes (Figure 1)
Summary
Psychrotolerant bacteria, predominant in cold stored raw milk, are known to secrete heat-resistant enzymes, which partly even withstand ultra-high temperature processing (von Neubeck et al, 2015; Glück et al, 2016). The quality of ultra-high temperature treated (UHT) products decreases and premature spoilage may occur, presenting considerable economic challenges for the milk industry (McKellar, 1981; Sørhaug and Stepaniak, 1997; Stoeckel et al, 2016). Apart from the cultivation temperature, other extrinsic and intrinsic factors such as the composition of the cultivation medium, iron content, quorum sensing, and phase variation were shown to influence peptidase activities of Pseudomonas members in a species- and partially strain-specific manner (Nicodeme et al, 2005; Maunsell et al, 2006; Liu et al, 2007; Alves et al, 2018). The complex regulatory processes behind these variations are not yet fully understood
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