List of strains and plasmids used in the present work.

<p>List of strains and plasmids used in the present work.</p

Inhibition of VvRraA1 and VvRraA1-C9D on the cleavage of p-BR10+hpT by VvRNase E <i>in vitro</i>.

<p>0.5 pmol of 5’-end-labeled p-BR10+hpT RNA was incubated with 1 pmol of VvRne with varying concentrations of VvRraA1 and VvRraA1-C9D, 50 pmol of VvRraA1, or 50 pmol of BSA in 20 μl of 1 × cleavage buffer at 37°C for 2 h for VvRne, VvRraA1 only, or BSA only controls. Samples were mixed with an equal volume of loading buffer, and then denatured at 65°C for 5 min and loaded onto a 12% polyacrylamide gel containing 8 M urea. The percentage of uncleaved p-BR10+hpT in the gel was quantitated using a phosphorimager and OptiQuant software.</p

Alignment of amino acid sequences of <i>E</i>. <i>coli</i> RraA and its orthologs in Gram-negative bacteria.

<p>(A) Alignment of amino acid sequence using CLUSTAL W. VvRraA1 and VvRraA2; Amino acid sequences of RraA homologs from <i>E</i>. <i>coli</i> (EcRraA), <i>Mycobacterium tuberculosis</i> (MtRraA), <i>P</i>. <i>aeruginosa</i> (PaRraA), <i>Vibrio cholerae</i> (VcRraA), <i>V</i>. <i>vulnificus</i> (VvRraA1 and VvRraA2) are used. Arrows indicate conserved Cys9 and Cys41 residues of RraA proteins. (B) A molecular model for the C9D mutant of <i>E</i>. <i>coli</i> RraA. The model of the mutant protein was built based on the wild-type structure of <i>E</i>. <i>coli</i> RraA (PDB code: 1Q5X). The subunits are displayed in different colors (cyan and yellow). The mutated Asp9 is positioned in the hydrophobic pocket lined with residues in gold at the interface between the two neighboring subunits, which would destabilize the oligomeric forms of the protein (left lower box). The near region of Cys9 structure is shown in the left upper box.</p

Oligomerization state of EcRraA protein.

<p>Size exclusion chromatography with MALS of EcRraA was performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190064#pone.0190064.g002" target="_blank">Fig 2</a>. <i>a</i>, 87.05 kDa (~hexamer).</p

Interactions of VvRNase E with VvRraA proteins.

<p>Hexahistidine-tagged VvRraA1, VvRraA1-C9D, VvRraA2, VvRraA2-C9D, and the GST-fused VvRne (612–816 residues) were expressed and purified as described in the Methods section. The GST-fused VvRne protein was bound to GSH resin and incubated with VvRraA proteins and their C9D mutant proteins. Then, the proteins were eluted and the fractions were analyzed using SDS-PAGE. The protein bands were stained with Coomassie blue. Only VvRraA1 could tightly bind to VvRne.</p

Oligomerization state of VvRraA proteins. Size exclusion chromatography with MALS of VvRraA proteins and their C9D mutants.

<p>The molecular sizes of the peaks were estimated by using MALS, as indicated. Based on these results, <i>a</i>, corresponds to 75.62 kDa (~hexamer); <i>b</i>, 16.3 kDa (monomer); <i>c</i>, 56.64 kDa (trimer); <i>d</i>, 19.68 kDa (monomer). Although the peak ‘<i>a</i>’ for the wild-type VvRraA1 is in between hexameric and trimeric sizes (~4.7 times higher than the monomeric mutant), it is most likely that VvRraA1 is a hexameric form since all the native RraA proteins are in the forms of a trimer or a hexamer.</p

List of primers used in the present study.

<p>List of primers used in the present study.</p

Effects of the C9D mutation on the activity of VvRraA proteins <i>in vivo</i>.

<p>(A) Effects of co-expression of VvRraA proteins on growth in <i>E</i>. <i>coli</i> cells overproducing VvRNase E. The cultures of DK001 cells containing pKAN6B, pKAN6B-VvRraA1, pKAN6B-VvRraA1-C9D, pKAN6B-VvRraA2, or pKAN6B-VvRraA2-C9D were grown in LB-10 μM IPTG medium and 0.2% arabinose and no additional IPTG (DK001+ pKAN6B+ 10 μM IPTG) or 1 mM IPTG (DK001+ pKAN6B+1 mM, DK001+ pKAN6B-VvRraA1, DK001+ pKAN6B-VvRraA1-C9D, DK001+ pKAN6B-VvRraA2, and DK001+ pKAN6B-VvRraA2-C9D) were added to the cultures at OD₆₀₀ = 0.1~0.2. Then, their growth was monitored by analyzing cell density (absorbance at 600 nm) at specific time intervals. (B) Effects of co-expression of VvRraA proteins on the steady-state level of <i>rpsO</i> mRNA in DK001. Total RNA was isolated from DK001 cells grown to an OD₆₀₀ of 1.5 in the same manner described in Fig 3A, and reverse transcription-polymerase chain reaction (RT-PCR) was performed. The relative abundance of each mRNA is shown at the bottom of the gels. Student’s t-test was used for comparisons with control using SAS version 9.2 (SAS Institute, Cary, NC, USA). The data were repeated three times and presented as mean ± SEM. ***<i>p</i> < 0.001.</p