Genome Sequencing of five Lacticaseibacillus Strains and Analysis of Type I and II Toxin-Antitoxin System Distribution

Abstract

The analysis of bacterial genomes is a potent tool to investigate the distribution of specific traits related to the ability of surviving in particular environments. Among the traits associated with the adaptation to hostile conditions, toxin-antitoxin (TA) systems have recently gained attention in lactic acid bacteria. In this work, genome sequences of Lacticaseibacillus strains of dairy origin were compared, focusing on the distribution of type I TA systems homologous to Lpt/RNAII and of the most common type II TA systems. A high number of TA systems have been identified spread in all the analyzed strains, with type I TA systems mainly located on plasmid DNA. The type II TA systems identified in these strains highlight the diversity of encoded toxins and antitoxins and their organization. This study opens future perspectives on the use of genomic data as a resource for the study of TA systems distribution and prevalence in microorganisms of industrial relevance.

Keywords: DinJ/YafQ; Lacticaseibacillus; Lpt toxin; MazEF; Phd/Doc; YefM/YoeB; genome sequencing; toxin-antitoxin systems.

Figure 1

Circular representations of the chromosomes of Lacticaseibacillus isolates. The outer grey circle represents the genome of the reference strain used for the alignment, the green circle represents the contigs aligned with the reference sequence, the red circle represents structural contig rearrangements compared to the reference sequence. Genome scale is in Kbp. Below each circular representation the RefSeq accession number for each type strain is reported (see Table S1 for details). For strain L. rhamnosus Lc705 plasmid pLC1 was included in the comparison (RefSeq: NC_013200.1). (a) L. rhamnosus 1019 vs. L. rhamnosus Lc705; (b) L. rhamnosus 1473 vs. L. rhamnosus Lc705; (c) L. paracasei 2306 vs. L. paracasei JCM8130; (d) L. paracasei 2333 vs. L. paracasei JCM8130; (e) L. paracasei 4186 vs. L. paracasei JCM8130.

Figure 2

Phylogenetic analysis of Lacticaseibacillus isolates: (a) phylogenetic tree constructed by using newly sequenced strains and 15 reference strains, as inferred from core gene sequence alignment. Sequences were aligned using Roary, RAxML was used to construct a maximum-likelihood phylogenetic tree; (b) description of the pan genome of the 20 strains belonging to Lacticaseibacillus genus; (c) Venn diagram showing the number of shared and unique genes among the newly sequenced strains of Lacticaseibacillus genus.

Figure 3

Multiple sequence alignment of the four different peptides identified in the analyzed strains. * 10 bp intervals along with the sequence length indicators

Figure 4

Distribution and organization of the identified type I TA systems. Toxins and antitoxins are indicated by T and A, respectively. Abbreviations in strain names are as follows: LP: L. paracasei, LR: L. rhamnosus. The arrows indicate transcription direction of T and A, their absence highlights the promoter Scheme 15. Lactocaseibacillus strains used as references for genome analysis (Table S1). By using the tBlastn program, one Lpt and two Lptlike2 were identified, while no Lptlike and Lptlike1 were found. The identified Lpt sequence is located on the plasmid pLBPC-2 of L. paracasei JCM 8130 and it was previously described in Folli et al. [17]. Lptlike2 sequences were found in L. paracasei JCM 8130 and in L. paracasei ATCC334 and the alignment of the nucleotide sequences encompassing their coding regions is shown in Figure S3 in comparison with Lptlike2 of L.paracasei 2333. Interestingly, it was not possible to predict convergently transcripted RNA molecules able to act as antitoxins in any of these sequences.

Figure 5

Distribution and organization of the identified type II TA systems. The left part of the figure summarizes the distribution of toxins (T), antitoxins (A) and number of TA systems not organized in pairs, that can be considered as “orphans” (O) in the Lacticaseibacillus isolates. The right part of the figure shows the organization of the identified TA systems in the strains. A superimposed number indicates that multiple copies were found in the genome. Abbreviations in strain names are as follows: LP: L. paracasei, LR: L. rhamnosus.

Figure 6

Multiple sequence alignments of all the toxins identified in Lacticaseibacillus strains compared with E. coli homologous sequences. (a) YafQ proteins, E.coli sequence accession number HAM8829092; (b) MazF proteins, E.coli sequence accession number WP_000254738; (c) YoeB proteins, E.coli sequence accession number WP_112934640; (d) Doc proteins, E.coli sequence accession number WP_001216045. The catalytic residues identified in E. coli proteins are boxed in red and the numbering of E. coli residues is reported. Abbreviations prior to strain names are: pa: L. paracasei, rh: L. rhamnosus. * 10 bp intervals along with the sequence length indicators