Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays

Abstract

Lactococcus lactis subsp. lactis NCDO 2118 was recently reported to alleviate colitis symptoms via its anti-inflammatory and immunomodulatory activities, which are exerted by exported proteins that are not produced by L. lactis subsp. lactis IL1403. Here, we used in vitro and in silico approaches to characterize the genomic structure, the safety aspects, and the immunomodulatory activity of this strain. Through comparative genomics, we identified genomic islands, phage regions, bile salt and acid stress resistance genes, bacteriocins, adhesion-related and antibiotic resistance genes, and genes encoding proteins that are putatively secreted, expressed in vitro and absent from IL1403. The high degree of similarity between all Lactococcus suggests that the Symbiotic Islands commonly shared by both NCDO 2118 and KF147 may be responsible for their close relationship and their adaptation to plants. The predicted bacteriocins may play an important role against the invasion of competing strains. The genes related to the acid and bile salt stresses may play important roles in gastrointestinal tract survival, whereas the adhesion proteins are important for persistence in the gut, culminating in the competitive exclusion of other bacteria. Finally, the five secreted and expressed proteins may be important targets for studies of new anti-inflammatory and immunomodulatory proteins. Altogether, the analyses performed here highlight the potential use of this strain as a target for the future development of probiotic foods.

Fig 1. 16S phylogenetic tree and genomic heatmap of Lactococcus genus.

The Streptococcus thermophilus LMD-9 (position 17) was added to root the tree. The species in comparison are distributed from 1 to 17 in the same order, both vertically and horizontally. The numbers in the heatmap show the percentage of similarity between the species, varying from yellow (low similarity) to green (high similarity), or from 40% to 100%, respectively. The heatmap and the phylogenetic tree were created with the software Gegenees and Mega (Neighbor-Joining method with 1000 bootstraps replicates), respectively.

Fig 2. Circular comparison of the Lactococcus genus using L. Lactis NCDO 2118 as a reference.

Each ring of the circle corresponds to a specific complete genome represented in the legend on the right. The similarity between species is represented by the intensity of the color. Darker colors represent higher similarities than bright ones. Deleted regions are represented by blank spaces inside the circles. (GEI = Genomic Island; MI = Metabolic Island; SI = Symbiotic Island; MSI = Miscellaneous Island, harboring both metabolic and symbiotic factors). Genomic islands and phage sequences were predicted with GIPSy and PHAST, respectively. The circular genomic comparisons were created with BRIG.

Fig 3. Regions of bacteriocins predicted with BAGEL in L. lactis NCDO 2118.

BAGEL predicted three putative bacteriocins, one of each class. (A) Putative bacteriocin/Class I predicted on orf010 (pseudogene) and nisZ was found with manual curation. (B) Putative bacteriocin/Class II predicted on orf027 (pseudogene). (C) Putative bacteriocin/Sactipeptidase predicted on orf011 (this region was not previously characterized in the L. lactis subsp. lactis NCDO 2118 genome). All putative bacteriocins were also identified in Bactibase.

Fig 4. Photomicrograph of L. lactis NCDO 2118.

The measurements of the membrane wall were performed with ImageJ software using images generated with electron microscopy with EM10A equipment (Zeiss). Top: magnification of 50,000 times; bottom: magnification of 100,000 times.