Fig 1.
rrnI-dependent expression of HspA in V. vulnificus MO6-24/O strains.
(A) Schematic representation of the allele-specific RT-PCR analysis analyzing the relative amounts of I-rRNA or G-rRNA. (B) The number of I-rRNA or G-rRNA amplicons and other rRNAs amplified from the cDNA of the MO6 WT, MO6+rrnG, and MO6+rrnI strains was determined by PCR using common and allele-specific primers. The cDNA was synthesized from rRNAs purified from crude ribosomes of these strains. PCR products were resolved on a 2% agarose gel. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test (ns, not significant; ****, P < 0.0001). (C) Sequence alignment and secondary structure prediction of hspA mRNAs in the V. vulnificus strains MO6-24/O and CMCP6. Sequences of the hspA genes from V. vulnificus strains MO6-24/O and CMCP6 were obtained from the NCBI database (NC_014965.1 and NC_004459.2, respectively) and aligned using ClustalW (https://www.genome.jp/tools-bin/clustalw). The putative transcriptional start site (TSS) and the first nucleotide of the hspA gene are indicated in yellow and red, respectively. (D) Predicted secondary structures of hspA mRNAs from V. vulnificus CMCP6 (left) or V. vulnificus MO6-24/O (right). Secondary structures were obtained using the M-fold program (http://unafold.rna.Albany.edu). (E) rrnI-dependent expression of HspA. Vibrio vulnificus MO6-24/O strains (WT, +rrnG, and +rrnI) were grown in LBS at 30°C until mid-log phase and harvested for western blot analysis of HspA proteins using polyclonal antibodies against HspA. (F) Effect of rrnI expression on heat shock susceptibility of V. vulnificus MO6-24/O strains (WT, +rrnG, and +rrnI, and ΔhspA). The number of CFUs of each V. vulnificus MO6-24/O strain grown at 30°C or transiently grown at 45°C for 180 min was measured. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test (ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 2.
Co-immunoprecipitation (co-IP) showing the predominant association of I-ribosomes with HspA nascent peptides in V.
vulnificus MO6-24/O strains. (A) Identification of HspA and the RNAP-β subunit after co-IP. The precipitate was subjected to western blotting with antibodies against HspA or RNAP-β. S1 protein was used as a loading control. (B) Characterization of the expression and assembly of I-rRNAs after co-IP. The number of amplicons of 23S I-rRNAs and other 23S rRNAs amplified from the cDNAs of the MO6 WT, MO6+rrnG, and MO6+rrnI strains was determined by RT-PCR using common and allele-specific primers. The cDNA was synthesized from rRNAs purified from immunoprecipitated samples or crude ribosome samples, which was used as controls. PCR products were resolved on a 2% agarose gel. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test (ns, not significant; ****, P < 0.0001).
Fig 3.
Effect of rrnI expression on the virulence of V.
vulnificus MO6-24/O in mice. (A) Plasmid loss rate of V. vulnificus MO6-24/O in mice. Pathogen-free 7-week-old female ICR mice that were pretreated with tetracycline and iron dextran (n = 5 mice per group) were subcutaneously injected with 1 × 106 cells of V. vulnificus MO6-24/O (MO6 WT). Mice with tetracycline administered orally before infection and those treated once more 6 h after the initial treatment were sacrificed 6 h and 12 h after infection, respectively. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined using a two-tailed unpaired Student’s t-test (ns, not significant; ****, P < 0.0001). (B) Survival rates and the survival time of mice infected with V. vulnificus MO6-24/O strains (MO6 WT, MO6+rrnG, and MO6+rrnI). Pathogen-free 7-week-old female ICR mice pretreated with iron dextran (n = 10 mice per group) were intraperitoneally injected with 8 × 102 cells of the V. vulnificus MO6-24/O strains. Survival rate was monitored for 25 h. Data are presented as the mean ± SEM of three independent experiments. Two-tailed unpaired Student’s t-tests were used to assess significant differences: ** denotes P < 0.01 for comparisons of MO6+rrnI-infected mice versus WT- or MO6+rrnG-infected mice. (C) Viable bacterial counts in the spleen and liver of mice infected with the V. vulnificus MO6-24/O strains (MO6 WT, MO6+rrnG, and MO6+rrnI). Six hours after bacterial infection, the mice (n = 5 mice per group) were sacrificed. Results are expressed as numbers of CFU/g of each organ. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test (ns, not significant; ****, P < 0.0001).
Fig 4.
rrnI-dependent expression of HspA in V. fischeri MJ11 strains.
(A) Sequences of hspA genes in V. fischeri MJ11 and V. vulnificus CMCP6 were obtained from the NCBI database (NC_011184.1 and NC_004459.2, respectively) and were aligned using ClustalW. The putative transcriptional start site of V. vulnificus CMCP6 and the first nucleotide of the hspA sequence are indicated in yellow and red, respectively. (B) Prediction of the secondary structures of V. fischeri MJ11 hspA mRNAs. Secondary structures were obtained using the M-fold program. (C) V. fischeri MJ11 strains (WT, +rrnG, and +rrnI) were grown and harvested for a western blot analysis of HspA proteins using polyclonal antibodies against HspA (see the Fig 1E legend for details). (D) Effect of rrnI expression on heat shock susceptibility of V. fischeri MJ11 strains (WT, +rrnG, and +rrnI). The number of CFUs of each V. fischeri MJ11 strain was measured as described in the legend of Fig 1F. The expression levels of HspA and the number of CFUs were compared by setting those of the WT to 1. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test (ns, not significant; *, P < 0.05 and ***, P < 0.001).
Fig 5.
The role of the I-ribosome in bacterial survival.
(A and B) Phylogenetic trees of rRNA operons in V. vulnificus strains. Neighbor-joining phylogenetic trees based on 16S rRNA (A) and 23S rRNA (B) sequences showing the positions of rRNA operons within the Vibrio genomes. Bold text represents the V. vulnificus CMCP6 rRNA operon. V. vulnificus strains that contain rRNA genes similar to the rrnI operon were selected using NCBI nucleotide blast (https://blast.ncbi.nlm.nih.gov) and the rRNA gene sequences of these strains were obtained from the NCBI database. The rRNA genes of each strain were named starting with “A” according to the nucleotide position in the genome. The values above and below the branches (expressed as percentages) indicate the robustness of the corresponding branch as determined by bootstrap analysis (heuristic search). (C) I-ribosomes from V. vulnificus CMCP6 were exogenously expressed in V. vulnificus MO6-24/O strains. Overexpression of rrnI in the V. vulnificus MO6-24/O strain leads to increased bacterial survival in heat shock and host environments by preferentially translating the target mRNAs.