Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus

Abstract

Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable "toxin", a protein or peptide capable of killing the cell upon plasmid loss and by an unstable "antitoxin", a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.

Figure 1

Electrophoretic patterns of cDNA-AFLP experiments conducted on L. rhamnosus strains PR1473 (a) and PR1019 (b) grown in MRS or CB medium. Lane M, IRDye700 sizing standard. Lanes 1, 3 and 5, MRS medium with the following primer combinations: lane 1, EcoRI-AC/MseI-AT; lane 3, EcoRI-AT/MseI-AC; lane 5, EcoRI-AT/MseI-AT. Lanes 2, 4 and 6, CB medium with the following primer combinations: lane 2, EcoRI-AC/MseI-AT; lane 4, EcoRI-AT/MseI-AC; lane 6, EcoRI-AT/MseI-AT. The overexpressed amplified fragments corresponding to the plasmid sequence are boxed and magnified on the right side of each gel. The full length images of the gels are reported in Fig. S1.

Figure 2

Structure of the putative toxin–antitoxin locus. (a) Alignment between the plasmid sequences overexpressed in L. rhamnosus PR1473 and PR1019 identified by cDNA-AFLP and homologous DNA sequence of plasmid pLBPC-2 from L. paracasei (GenBank accession number AP012543 range 2009–1541). The predicted elements of the identified TA locus are: black lines, RNA I and RNA II promoters and corresponding transcription terminators (TT); dashed black boxes, direct repeat regions (DR); black arrows, start and stop codons of the toxin peptide; gray boxes, primers TA-plus and TA-minus employed to amplify the complete TA system region. (b) Schematic representation of the Lpt TA locus showing the two convergently transcribed RNA I and RNA II (black arrows) and the corresponding promoters (P boxes). The shaded box represents the toxin coding region with the peptide sequence written above. (c) Secondary structures of RNA I (toxin) and RNA II (antitoxin) predicted with the RNAfold WebServer. The 5′ end of the toxin RNA identified by RACE experiments is indicated with an arrow. DR, direct repeat region; UH, upstream helix; SL stem-loop. On toxin and antitoxin RNAs, dots indicate DR sequences. On toxin RNA black boxes indicate the RBS region and white dots the Lpt start codon. RNA nucleotides are colored by base-pairing or unpairing probabilities.

Figure 3

Characterization of L. rhamnosus RNA I and RNA II sequence elements. (a) Sequence alignment of the TA region amplified from L. rhamnosus PR1473, L. rhamnosus PR1019 and the homologous sequence of L. paracasei pLBPC-2 plasmid. TA-plus and TA-minus primers used in PCR (black boxes); predicted RNA I and RNA II transcripts (black arrows); experimentally determined RNA I transcript (red arrow); predicted promoter regions (black dashed lines); start and stop codons of the toxin-coding region on RNA I (red boxes); sequence-specific primers used in 3′ RACE (blue line) and 5′ RACE (green line) experiments. (b) L. rhamnosus PR1019 sequencing profile of the amplified fragment obtained by 5′ RACE experiments. The 5′ end of the toxin RNA is in uppercase characters, the primer sequence is in lowercase characters and the black arrow points to the 5′ end nucleotide (see also Fig. S3). (c) L. rhamnosus PR1473 sequencing profile of the amplified fragment obtained by 3′ RACE experiments. The 3′ end of the toxin RNA is in uppercase characters.

Figure 4

Detection of RNA I and RNA II by qRT PCR. (a) Absolute quantification of RNA I and RNA II in L. rhamnosus PR1019. MRS medium (black bars); CB medium (grey bars). (b) Absolute quantification of RNA I and RNA II in L. rhamnosus PR1473. MRS medium (black bars); CB medium (grey bars). The error bars represent the standard deviation of the mean value from three independent experiments. (c) Summary table with RNA I and RNA II copy number values and corresponding ratio obtained for each strain in MRS and CB media.

Figure 5

Lpt toxicity in E. coli. Growth assays of E. coli DH10bT1R transformed with the empty vector pSRKKm (circles) or with the recombinant pSRKKm-lpt vector (triangles), in LB medium supplemented with lactose (black symbols) or glucose (white symbols).

Figure 6

Promoter mapping by atomic force microscopy. (a) Schematic representation of the 1065 bp long DNA fragment used in AFM experiments. Position of the promoters from the DNA ends is given with respect to the centre of the −10 hexamer. (b–d) Distribution of the RNAP position along the DNA template determined by contour length measurements of RNAP-DNA complexes imaged by AFM. The DNA template used is schematized at the top of each panel and representative complexes are shown on the right side. (b) wt RNA I and wt RNA II promoters; (c) inactivated RNA I and wt RNA II promoters; (d) wt RNA I and inactivated RNA II promoters. (e) Growth assays of E.coli XL1 Blue transformed with pGEM-TA harboring wt RNA I and wt RNA II promoters (black circles), inactivated RNA I and wt RNA II promoters (white circles) and wt RNA I and inactivated RNA II promoters (grey circles).

Figure 7

Alignment of TA sequence elements homologous to the lpt locus identified in plasmids of Lactobacillus genus (a) Representative sequence alignment of the putative TA systems. RNA I and RNA II promoters (black lines); direct repeat region DR (dashed black line); start codon, stop codon and RBS of RNA I (red boxes). Accession numbers and corresponding sequence intervals of the aligned TA loci are reported in Table 1. (b) Alignment of Lpt homologous peptides in Lactobacillus genus showing six different toxin peptides (numbered boxes) compared with the Fst toxin encoded by plasmid pAD1 of E. faecalis. Hydrophobic residues are marked with stars.