Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera

Abstract

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

Figure 1. Cladogram of 452 genera.

Cladogram of 452 genera from 26 phyla with the 213 genomes analysed in this study, based on the amino-acid sequences of 16 marker genes. The tree was built using the maximum likelihood method but visualized by removing the branch length information. The coloured branches indicate different genera sequenced in this research; grey branches indicate members of genera whose genomes were previously sequenced. The colours in the outer circle represent the phyla that are indicated in the legend, and the different shapes near branch-tips indicate the position of genera that are most closely related to Atopobium, Carnobacterium, Kandleria and Lactococcus, separately.

Figure 2. Maximum likelihood phylogeny derived from 73 core genes across 213 strains.

The phylogeny was estimated using the PROTCATWAG model in RAxML and rooted using the branch leading to Atopobium minutum DSM 20586, Olsenella uli DSM 7084 and Atopobium rimae DSM 7090 as the outgroup. Bootstrapping was carried out using 100 replicates and values are indicated on the nodes. Colours on taxon labels indicated the presence of CRISPR-Cas systems using blue, red and green for Type I, II and III systems, respectively. Undefined systems are represented in orange. Colour combinations were used when multiple systems from different families were concurrently detected in bacterial genomes.

Figure 3. Heat map illustrating the distribution and abundance of glycoside hydrolase (GH) family genes across the Lactobacillus Genus Complex and associated genera.

Gene copy number of each of the 48 represented GH families is indicated by the colour key ranging from black (absent) to green. Strains are graphed in the same order left to right as they appear top to bottom in the phylogeny (Fig. 2) with the isolation source of each strain indicated by the colour bar at the top of the heat map.

Figure 4. Differential abundance of genes encoding LPXTG proteins, sortases, pili and cell envelope proteases (a–d, respectively).

The y axis indicates the number of genes/clusters detected. Strains are graphed in the same order left to right as they appear top to bottom in the phylogeny (Fig. 2). In c, each black bar indicates strains belonging to the same lineages. (c) Labels: i, the L. composti clade; ii, the L. casei/rhamnosus clade; iii, the L. ruminis clade; iv, the L. brevis/parabrevis clade; v, the Pediococcus ethanolidurans clade. (d) Labels: LX, LPXTG-sortase-dependent anchor (including derivatives); S, S-layer type anchor; T, truncated protein.

Figure 5. Comparative analysis of CRISPR sequences.

The tree in a is derived from an alignment of the sequence of the universal Cas protein, Cas1, to create a phylogenetic tree based on the relatedness of all CRISPR-Cas systems in lactobacilli and closely related organisms. Types I, II and III are represented in blue, red and green, respectively. The tree in b is derived from an alignment of Cas9, the signature protein for Type II systems, to create a phylogenetic tree showing the relatedness of Cas9 proteins from Type II-A and II-C systems identified in lactobacilli and closely related organisms. A subset of short Type II-A Cas9 proteins is highlighted. In c, key guide sequences driving DNA targeting by Cas9 are shown for L. jensenii, L. buchneri and L. mali. Predicted crRNA and tracrRNA sequences are shown at the top (red). Complementarity between CRISPR spacer sequences and target protospacer sequences (blue) in target nucleic acids is shown for phages and plasmids. The predicted protospacer-adjacent motif (PAM) sequences flanking the 3′ end of the protospacer sequence are shown in green.