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

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.

PCR primer, Table S1). Finally, the specific cDNA sequence was amplified by using touch-down PCR, the 5RACE PCR primer 1 µM and the reverse gene-specific primer 5RACE-minus 3 µM. The amplification products were then cloned into pGEM vector (Promega) and recombinant plasmids were sequenced on both strands.
Quantitative reverse transcription PCR qRT PCR was carried out using QuantStudio® 3 (Thermo Fisher Scientific), Power SYBR Green PCR Master Mix (Applied Biosystems) and specific primers as reported in Table S2. The 20 µl PCR reactions were incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Threshold cycle (Ct) data were determined using the default threshold settings and mean Ct values were determined from three PCR replicates.
Absolute quantification of toxin and antitoxin cDNAs from L.rhamnosus PR1019 and PR1473 was achieved by using a strain-specific calibration curve of Ct values obtained with increasing amounts of the lptcontaining DNA amplified by PCR with primers TA-plus and TA-minus (Table S1). Slope, efficiency and confidence values of the calibration curve obtained for each specific target and primer combination, are reported in Table S2. ANOVA followed by Tukey's test was applied to compare toxin/antitoxin cDNAs ratios calculated under the different experimental conditions. In the case of Parmigiano Reggiano cheese samples, relative toxin cDNA quantification was calculated using the 2− ∆∆CT method 47 , and 16s rRNA was used as internal standard. Statistical significance was determined by means of the Student's t-test for pairwise comparisons. All statistical analysis were performed using IBM SPSS v.23.
Transcription complex assembly and AFM imaging Promoter complexes were obtained by mixing 25 nM DNA with 70 nM E.coli RNAP holoenzyme (New England Biolabs) in transcription buffer (20 mM Tris-HCl pH 7.9, 50 mM KCl, 5 mM MgCl 2 , 1 mM DTT) and incubated for 30 min at 37 °C. The reaction was diluted 10X in deposition buffer (4 mM HEPES pH 7.4, 10 mM NaCl, 2 mM MgCl 2 ) and deposited onto freshly-cleaved mica for 90 seconds. Afterwards, the mica disk was rinsed with MilliQ water and dried with a gentle nitrogen flow. AFM imaging was performed in air with the tapping mode using a Nanoscope IIIA microscope (Veeco) equipped with E scanner and NSC14/Al BS commercial silicon cantilevers (MikroMasch). Images of 512×512 pixels were collected with a scan size of 2 µm at a scan rate of 2.5 lines per second.
DNA contour length measurements were performed as described in 47 using the following contour length estimator: L = (0.963ne + 1.362no) x S/W, where "ne" and "no" are the number of even and odd chain codes respectively, S is the image scan size, W is the image width in pixels. Position of the DNA bound RNAP was manually selected by clicking with the mouse the center of the protein globular feature. The DNA contour length from the protein to the nearest end was defined short-arm while the DNA contour length from the protein to the farther end was defined long-arm.