Comparative Genomics Analysis of Lactobacillus ruminis from Different Niches

Abstract

Lactobacillus ruminis is a commensal motile lactic acid bacterium living in the intestinal tract of humans and animals. Although a few genomes of L. ruminis were published, most of them were animal derived. To explore the genetic diversity and potential niche-specific adaptation changes of L. ruminis, in the current work, draft genomes of 81 L. ruminis strains isolated from human, bovine, piglet, and other animals were sequenced, and comparative genomic analysis was performed. The genome size and GC content of L. ruminis on average were 2.16 Mb and 43.65%, respectively. Both the origin and the sampling distance of these strains had a great influence on the phylogenetic relationship. For carbohydrate utilization, the human-derived L. ruminis strains had a higher consistency in the utilization of carbon source compared to the animal-derived strains. L. ruminis mainly increased the competitiveness of niches by producing class II bacteriocins. The type of clustered regularly interspaced short palindromic repeats /CRISPR-associated (CRISPR/Cas) system presented in L. ruminis was mainly subtype IIA. The diversity of CRISPR/Cas locus depended on the high denaturation of spacer number and sequence, although cas1 protein was relatively conservative. The genetic differences in those newly sequenced L. ruminis strains highlighted the gene gains and losses attributed to niche adaptations.

Keywords: CRISPR/Cas; Lactobacillus ruminis; bacteriocins; carbohydrate utilization; phylogenetic relationship; prophage.

Figure 1

Pan-genome and core-genome of L. ruminis. (a) The pan-genome represented by the accumulated number of new genes against the number of genomes added. The core-genome represented by the accumulated number of genes attributed to the core-genomes against the number of added genomes. The mathematical functions of the pan- and core-genome based on 91 strains of L. ruminis are also shown on the graph; (b) Venn diagram of homologous genes of L. ruminis.

Figure 2

Phylogenetic analyses of L. ruminis. (a) The phylogenetic tree of L. ruminis based on orthologous genes. Unrooted phylogenies of the L. ruminis genomes were based on the multiple sequence alignment of core proteins and constructed with the neighbor-joining tree-building algorithm. Phylogenetic groups were highlighted in different colors. The text background color represented the source. Blue: horse; yellow: piglet; green: cow; red: human; purple: dog; orange: milk. Yellow circle: A region; green circle: B region; red circle: C region. The outer circle was divided into clade A–E, which was present with different colors and letters. (b) Source distribution map of isolating strains. According to the distance of strain source more than 1000 km, it can be divided into three regions. Each sampling point was labelled with different number. 1: Kashi; 2: Wusu; 3: Shawan; 4: Changji; 5: Dazi; 6: Zhangye; 7: Yongchang; 8: Xining; 9: Ruo’ergai; 10: Pengshan; 11: Lijiang; 12: Fengqiu; 13: Xiayi; 14: Bozhou; 15: Poyang.

Figure 3

A heatmap based on average nucleotide identity (ANI) value of L.ruminis.

Figure 4

The fermentation profiles of L. ruminis. (a) Fermentation ability was indicated in blue for positive, while yellow for negative; (b) predicted glycosyl hydrolase (GH)-encoding gene content of L. ruminis.

Figure 5

The carbohydrate utilization operon in L. ruminis. (A) lactose; (B) sucrose; (C) fructooligosaccharide (FOS); (D): raffinose.

Figure 6

Relative frequency of conserved sequences from classII bacteriocin in L. ruminis.

Figure 7

Bacteriocin gene operon in L. ruminis.

Figure 8

Phylogenetic analysis of clustered regularly interspaced short palindromic repeats /CRISPR-associated (CRISPR/Cas) system. (a) Phylogenetic tree of direct repeats in L. ruminis; (b) the CRISPR/Cas classification onto the phylogenetic tree of Cas1.

Figure 9

Secondary structure of repeats in L. ruminis.

Figure 10

Correlation between the number of spacer and prophage in L. ruminis. Mapping the number of spacers in each strain to the corresponding amount of prophage (p = 0.0241).