Evolution and Competitive Struggles of Lactiplantibacillus plantarum under Different Oxygen Contents

Abstract

Lactiplantibacillus (Lb.) plantarum is known as a benign bacterium found in various habitats, including the intestines of animals and fermented foods. Since animal intestines lack oxygen, while fermented foods provide a limited or more oxygen environment, this study aimed to investigate whether there were genetic differences in the growth of Lb. plantarum under aerobic vs. anaerobic conditions. Genomic analysis of Lb. plantarum obtained from five sources-animals, dairy products, fermented meat, fermented vegetables, and humans-was conducted. The analysis included not only an examination of oxygen-utilizing genes but also a comparative pan-genomic analysis to investigate evolutionary relationships between genomes. The ancestral gene analysis of the evolutionary pathway classified Lb. plantarum into groups A and B, with group A further subdivided into A1 and A2. It was confirmed that group A1 does not possess the narGHIJ operon, which is necessary for energy production under limited oxygen conditions. Additionally, it was found that group A1 has experienced more gene acquisition and loss compared to groups A2 and B. Despite an initial assumption that there would be genetic distinctions based on the origin (aerobic or anaerobic conditions), it was observed that such differentiation could not be attributed to the origin. However, the evolutionary process indicated that the loss of genes related to nitrate metabolism was essential in anaerobic or limited oxygen conditions, contrary to the initial hypothesis.

Keywords: Lactiplantibacillus plantarum; core-genome; nitrate metabolism; pan-genome.

Figure 1

Comparison of functional categories of 25 Lb. plantarum genomes based on COG (A) and SEED (B). Genome sequences of 25 Lb. plantarum strains originated from five origins were uploaded to COG and SEED viewer servers independently. Functional roles of annotated genes were assigned and grouped in subsystem feature categories. The length of the colored bar represents the number of genes assigned to each category, and the color represents the origin.

Figure 2

Sizes of core- and pan-genomes of 25 Lb. plantarum strains. Red curve (core genome) and blue curve (pan-genome) were fitted to decay function (937.967exp(−x / 11.490) + 1700.174) and Heap’s law function (2798.409 × 0.207), respectively. Each dot shows gene cluster number of an individual genome.

Figure 3

Venn diagrams of five Lb. plantarum genomes of different origins (A) or the same origins (B). Venn diagram generated using EDGAR. Overlapping regions represent Lb. plantarum CDSs shared between Lb. plantarum genomes. Numbers outside overlapping regions indicate numbers of CDSs in each genome without homologs in other sequenced Lb. plantarum genomes. Red, yellow, green, blue, and purple indicated animal, dairy, fermented meat, fermented vegetables, and human-derived strains.

Figure 4

Analysis of ancestral genes in evolutionary path. Numbers adjacent to internal nodes indicate the number of estimated ancestral genes (protein-coding genes). Right panel indicates the number of CDSs of Lb. plantarum strains.

Figure 5

Minimal gene gain and loss events under the best-fit model (GD-FR-ML). Numbers on branches denote the minimum number of gains and losses in that order.

Figure 6

Predicted hetero-lactic fermentative pathways of 25 Lb. plantarum strains under anaerobic conditions (A) and aerobic conditions (B). Enzyme-encoding genes and E.C. number are displayed in green. Metabolites are shown in orange box. Gene possession was marked with a box of colors corresponding to each strain.

Figure 7

Predicted respiratory chain (A), nitrate reduction (B), and reactive oxidation species persistence system (C) of 25 Lb. plantarum strains under anaerobic conditions (A) and aerobic conditions (B). Enzyme-encoding genes and E.C. number are displayed in green. Gene possession was marked with a box of colors corresponding to each strain.