Genomic Analysis of Three Cheese-Borne Pseudomonas lactis with Biofilm and Spoilage-Associated Behavior

Abstract

Psychrotrophic pseudomonads cause spoilage of cold fresh cheeses and their shelf-life reduction. Three cheese-borne Pseudomonas sp., ITEM 17295, ITEM 17298, and ITEM 17299 strains, previously isolated from mozzarella cheese, revealed distinctive spoilage traits based on molecular determinants requiring further investigations. Genomic indexes (ANI, isDDH), MLST-based phylogeny of four housekeeping genes (16S rRNA, gyrB, rpoB and rpoD) and genome-based phylogeny reclassified them as Pseudomonas lactis. Each strain showed distinctive phenotypic traits at 15 and 30 °C: ITEM 17298 was the highest biofilm producer at both temperatures, whilst ITEM 17295 and ITEM 17299 showed the strongest proteolytic activity at 30 °C. A wider pattern of pigments was found for ITEM 17298, while ITEM 17295 colonies were not pigmented. Although the high genomic similarity, some relevant molecular differences supported this phenotypic diversity: ITEM 17295, producing low biofilm amount, missed the pel operon involved in EPS synthesis and the biofilm-related Toxin-Antitoxin systems (mqsR/mqsA, chpB/chpS); pvdS, required for the pyoverdine synthesis, was a truncated gene in ITEM 17295, harboring, instead, a second aprA involved in milk proteolysis. This work provided new insight into the food spoiler microbiota by identifying these mozzarella cheese spoilers as P. lactis; molecular targets to be exploited in the development of novel preservative strategies were also revealed.

Recent Advances in the Mechanisms and Regulation of QS in Dairy Spoilage by Pseudomonas spp.

“…As a novel species, little information is reported regarding the QS by these strains. This species harbours genetic determinants for Las, LuxR, and RhlI (synonymous BjaI) required for the production of N -butanoyl-L-homoserine lactone and N -hexanoyl-L-homoserine lactone [ 35 ]. In P. aeruginosa las and rhl QS systems operate in a hierarchy wherein the N -3-oxododecanoyl-homoserine lactone, a “gatekeeping” signalling molecule of the las system, regulates the rhl system at the transcriptional and posttranslational levels [ 123 ].…”

“…In P. aeruginosa las and rhl QS systems operate in a hierarchy wherein the N -3-oxododecanoyl-homoserine lactone, a “gatekeeping” signalling molecule of the las system, regulates the rhl system at the transcriptional and posttranslational levels [ 123 ]. By the activation of a cascade of signals, including the RsmA/RsmZ, the two-component system GacA/GacS, responsible for sensing and responding to environmental stimuli, positively controls the QS operon comprising las and rhl ; in P. lactis ITEM 17298 GacA/GacS were described as BarA/UvrY [ 9 , 35 ].…”

“…Other QS regulators, such as the acyl-homoserine-lactone synthase EsaR, and the HTH-type QS-dependent transcriptional regulator RpaR, responding to the autoinducer 4-coumaroyl-homoserine lactone, were identified in P. lactis isolated from fresh cheeses [ 9 , 35 ]. In P. aeruginosa EsaR is an unusual example, as it binds the target DNA in the absence of a signal and serves as a repressor of QS [ 124 ].…”

“…In milk, the AHLs signal molecules (C4-HSL and 3OC8-HSL) from Pseudomonas species, such as P. azotoformans , were found to change their particle size distribution, and lead to the destabilization of UHT milk during storage; this feature, as well as off taste and off odour, was putatively attributed to the production of QS-regulated heat-resistant proteases and lipases produced by psychrotrophic bacteria [ 13 , 137 ]. Indeed, AHLs and other AHL-related products from Pseudomonas species have been shown to increase the activity of the apr X promoter; the activity was instead repressed in presence of the enzyme AHL lactonase hydrolysing AHLs [ 35 , 57 ]. Post-transcriptional regulation, under the control of the GacS/GacA two-component regulatory system, was also suggested to take place in the expression and secretion of protease in this system [ 57 ].…”

“…Few studies report the regulation of lipase genes and include P. aeruginosa; in this strain the expression of the lipase genes was controlled by the RhlR/I system and Gac system [ 141 ]. In P. lactis and P. fluorescens [ 35 , 138 ], isolated by dairy products, the activity of lipase included in apr X- lip A operon were by the homologue of the E. coli env Z- omp R affected by environmental osmolarity and regulating biofilm formation [ 142 ].…”