In this study, comparative genomics analysis was done on the whole genome sequence of five strains of L. pontis from different sources. It was found that L. pontis has a smaller genome and a higher G + C% content than a lot of LAB. The length of genome sequence of different LAB is between 1.80 and 2.90 Mb, and the genome size of L. pontis is within that range. The GC content of most LAB genomes is below 50%, the GC content of Ligilactobacillus salivarius UCC118(Netherlands 2008) is at least 33%. The GC content of L. pontis was higher than a lot of LAB. In bacteria a high GC content is related to a faster growth rate and a more stable DNA structure (Bobay and Ochman 2017); not only is the genome density relatively large, but also the ability to resist high temperature and alkaline environment is enhanced (Musto et al. 2004). Yue Xiao (Xiao et al. 2021) observed a reduction in the GC content in host-adapted Lactobacillus species compared with nomadic species and free-living species, this is contrary to the results of this study.
Based on the core-genome phylogenetic tree, it was found that isolates from fermented milk, sourdough and chicken gastrointestinal tract were in different branches; the branch that included isolates from chicken gastrointestinal tract was closer to the common ancestor. Conclusions from ANI and TNI analysis were consistent with those from phylogenetic tree analysis. The ANI values of IMAU10341 and IMAU10345 waere high and different to the ANI values of the type strains. The two strains isolated from similar samples were closely related, but not identical. Strains from fermented milk, sourdough and broiler chicken gastrointestinal tract naturally clustered into several categories. In addition, there were some differences in ANI values between strains from different sources and the type strain, and the ANI values of L. pontis strains from different hosts indicate that the species have habitat adaptability.
In this study, the number of genes specific to a strain were similar when strains were from the same source, but differed greatly when strains were from different sources. The unique genes of L. pontis were under positive selection to adapt to different environments, and this study demonstrated that food and intestinal habitats exerted different selective pressures that were related to growth rate and metabolism (Maldonado-Gómez et al. 2016). The unique genes of IMAU10341 and IMAU10345 were pseudogenes. Some original genes can be degraded or inactivated if they are no longer needed. The formation of pseudogenes is a mechanism for gene decline or deletion in the genome (Lerat 2005). We speculate that fermented milk is nutritious and suitable environment, which leads to the phenomenon of genome degradation in strains compared with strains from other environments.
Shuo Wang, et al., (Wang et al. 2020) evaluated the phylogenetic relationships amongst L. ruminis strains and created a phylogenetic tree based on orthologous genes from 91 genomes that constituted the core-genome. They found that strains from porcine, bovine and human faeces were divided into three clades; from the phylogenetic tree strains from the same sample type were clustered, closely related, and with the potential for niche adaptation. In our study, MAG.251 was closest to the root, indicating that its genome is closer to its ancestors than the other strains evaluated. In different environments, the evolutionary process of species adaptation is different. Adaptation of the bacterial genome to specific environments will accumulate genetic variation related to those environmental characteristics. The genomes of the other four strains all changed due to environmental changes. Strains from different sources clustered together, which indicated that the phylogenetic relationship reflected potential niches adaptation which was consistent with ANI results. Adaptive evolution of microorganisms to specific niches is also reflected in their functional genomics, and specific niches will shape specific functional genomics. In order to reduce reproduction-related energy consumption, strains living in specific niches will lose redundant functional genes, and at the same time evolve specific genes adapted to specific niches (Jung et al. 2018).Functional gene annotation results found that L. pontis isolates from fermented milk (IMAU10341 and IMAU10345) were rich in carbohydrate-related genes and genes involved in amino acids and derivatives. Genes related to metabolism of fatty acids, lipids and isoprenoids were significantly more abundant in fermented milk isolates than in other strains, while genes for protein metabolism were more abundant in the sourdough isolate LP475 than in other strains.
Based on RAST annotations, carbohydrates are the main energy source for growth and development of organisms. Studies have shown that LAB can specifically adapt to the environment by obtaining carbohydrate-related genes from the environment or losing non-essential carbohydrate genes. Differences in carbohydrate genes amongst the five L. pontis strains may be related to their different isolation sources; strains from the same isolation source were very similar. Those isolated from fermented milk had more cell wall and capsule genes than other strains. This may be due to the rich environment of fermented milk that also contains many other microorganisms; increasing the thickness of the cell wall would help resist external antibacterial substances.
LAB require exogenous amino acids and peptides which are provided by hydrolysis of casein in fermented milk. Many LAB are deficient in a variety of amino acids (Walter et al. 2011). In addition to enabling microbial growth, peptides, amino acids and their derivatives contribute to the formation of the unique texture and flavour of fermented milk, so strains from fermented milk contain more genes associated with metabolism of amino acids and their derivatives.
In addition, all L. pontis strains contained genes for producing extracellular polysaccharides, which protect them against external stresses. Because L. pontis contains more genes associated with carbohydrate metabolism, we also undertook carbohydrate enzyme analysis. CAZY annotation showed that the distribution and abundance of CE, GH, and GT families of genes in L. pontis were visualized by heat map and clustered using a hierarchical clustering method. This found that L. pontis was divided into three groups, representing the three different isolation sources. There are differences in the composition of carbohydrate active enzymes. The strains of L. pontis contained eight GT families, amongst which GT2 and GT4 were the most widely distributed. GT2 is a diverse family. Either the sugar is changed from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose, or CDP-varying sugars are transferred to a range of substrates including cellulose, dihydroethanol phosphate and phosphoric acid. Studies have shown that GT2 and GT4 are rich in glycosyltransferases in naturally fermented mares’ milk. In fermented milk, these substances, especially polysaccharide compounds, may be related to the final viscosity and water holding capacity of fermented milk products. The L. pontis strains were rich in GT2 and GT4, indicating that they may have higher sugar synthesis ability. In addition, the GT32 family, which participates in conversion of mannose, glucose and galactose, and the GT101 family, which represents β-glucosyltransferase, were only present in MAG.251; this may be related to its animal origin. GH2 mainly encodes β-glycosidases, these enzymes are usually related to metabolism of lactose and galactose, GH13 mainly encodes α-glycosidases, which are related to the metabolism of starch, and GH25 and GH73 mainly encode lysozymes, which are related to antibacterial activities of bacteria (originally found in fungi or bacteriophages). This kind of family has a large number of strains from herbivores, kefir and porridge. The possible reasons for this is that the genetic changes made by the strains to resist external environmental factors indicate that they can resist external change. GH53 was only found in sourdough isolates, which may be related to its ability to metabolise hemicellulose (Douillard et al. 2013).
Bacteriocins are bacteriostatic substances produced by LAB in order to compete for ecological niches. Amongst the five strains of L. pontis, four that were derived from fermented milk, sourdough and broiler chicken gastrointestinal tract, produced a lot of bacteriocins. IMAU10341, IMAU10345 and DSM8475T produced nisin which is a polypeptide that, together with acids and peroxides, is among the biological inhibitors produced by Lactococcus lactis. One potential function of nisin is that it has a regulatory action on the growth cycle of the producing organism (Ruyter et al. 1996), enabling them to compete under real-life conditions (Wang et al. 2020). Results from functional genome analysis were the same as the phylogeny results, and gene expression of the different strains was similar, reflecting potential niche adaptation. At present, there are few published genomes for L. pontis which limits in-depth study of the species, and it may be necessary to further explore appropriate culture conditions, increase the number of strains isolated from various sources and samples for research.
The results of this study provide a reference for understanding the evolution of L. pontis. Phylogenetic and functional genomic characteristics prove that L. pontis strains have potential niche adaptations. However, due to the limited number of strains available, further comparative genomic research is needed for verification.