Enzymatic activity necessary to restore the lethality due to Escherichia coli RNase E deficiency is distributed among bacteria lacking RNase E homologues

Abstract

Escherichia coli RNase E (Eco-RNase E), encoded by rne (Eco-rne), is considered the global RNA decay initiator. Although Eco-RNase E is an essential gene product in E. coli, some bacterial species, such as Bacillus subtilis, do not possess Eco-RNase E sequence homologues. B. subtilis instead possesses RNase J1/J2 (Bsu-RNase J1/J2) and RNase Y (Bsu-RNase Y) to execute RNA decay. Here we found that E. coli lacking the Eco-rne gene (Δrne E. coli) was viable conditional on M9 minimal media by introducing Bsu-RNase J1/J2 or Bsu-RNase Y. We also cloned an extremely short Eco-RNase E homologue (Wpi-RNase E) and a canonical sized Bsu-RNase J1/J2 homologue (Wpi-RNase J) from Wolbachia pipientis, an α-proteobacterial endosymbiont of arthropods. We found that Wpi-RNase J restored the colony-forming ability (CFA) of Δrne E. coli, whereas Wpi-RNase E did not. Unexpectedly, Wpi-RNase E restored defective CFA due to lack of Eco-RNase G, a paralogue of Eco-RNase E. Our results indicate that bacterial species that lack Eco-RNase E homologues or bacterial species that possess Eco-RNase E homologues which lack Eco-RNase E-like activities have a modest Eco-RNase E-like function using RNase J and/or RNase Y. These results suggest that Eco-RNase E-like activities might distribute among a wide array of bacteria and that functions of RNases may have changed dynamically during evolutionary divergence of bacterial lineages.

Fig 1. Molecular phylogenetic tree of RNase E/G family proteins derived from 15 representative bacterial lineages.

The evolutionary history was inferred using the maximum likelihood method (see the Materials and methods for details). The percentage of trees in which the associated taxa clustered together (bootstrap values) is shown next to the branches. The tree is drawn to scale and the branch lengths were operationalised as number of substitutions per site. Nodes with less than 50% bootstrap support are collapsed. KEGG organism codes are given in parentheses. NCBI Protein IDs are given in square brackets. Superscripts E and G represent those that were registered on the database as RNase E and RNase G, respectively. Highly supported clades containing E. coli RNase E and RNase G are highlighted by shading.

Fig 2. Growth of RNase E-, RNase J-, or RNase Y-complemented Δrne E. coli strains on LB and M9 plates with various carbon sources.

Cultures of MT1282 (a), MT696 (b), MT1072 (c), MT1070 (d), MT1125 (e), MT928 (f), and MT956 (g) were spread on LB or M9 gellan gum plates with various carbon sources containing (0.1% ara) or lacking [ara(-)] 0.1% L-(+)-arabinose, as indicated. Appropriate concentrations of IPTG (10 μM for MT928, MT956, and MT1125; 50 μM for MT1072; no IPTG (leaky expression) for MT696, MT1070, and MT1282) were added to the plates. Plates were scanned after incubation at 37°C for 6 days. Glyce glycerol, Pyru pyruvate.

Fig 3. Morphology of Δrne E. coli cells complemented by Bsu-RNase J1, Bsu-RNase Y, or Wpi-RNase J.

Single colonies of the parental MG1655 strain (Wild-type) (a), MT1278 (Bsu-RNase J1) (b), MT1254 (Bsu-RNase Y) (c), and MT1094 (Wpi-RNase J) (d) grown on M9-glycerol plates (0.6% gellan gum) were suspended in M9 minimal medium and then spread directly onto glass slides. Slides were prepared for microscopic observations, as previously described [25], except that poly-L-lysine was omitted. Cells were stained with DAPI (ProLong Diamond Antifade Mountant with DAPI) and microscopic images were obtained using an Axiovert 200 system (Zeiss). A scale bar measuring 5 μm is shown below each DIC image.

Fig 4. Effects of endogenous Eco-RNase G on the restoration of CFA in Bsu-RNase Y-, Bsu-RNase J1/J2-, or Wpi-RNase J-complemented Δrne E. coli.

Cultures of MT1177 (a), MT1158 (b), MT1176 (c), or MT1173 (d) were spread onto LB and M9 plates (0.6% gellan gum) with various carbon sources containing (0.1% ara) or lacking [ara(-)] 0.1% L-(+)-arabinose, as indicated. Appropriate concentrations of IPTG (10 μM for MT1173 and MT1176; 50 μM for MT1177; no IPTG (leaky expression) for MT1158) were added to the plates. Plates were scanned after incubation at 37°C for 14 days. Glyce glycerol, Pyru pyruvate.

Fig 5. Effects of the ribonucleolytic activity on the restoration of CFA in Δrne E. coli.

Cultures of MT1288 (a), MT1315 (b), MT1200 (c), MT1266 (d), or MT983 (e) were spread onto LB or M9 plates (0.6% gellan gum) with various carbon sources containing (0.1% ara) or lacking [ara(-)] 0.1% L-(+)-arabinose, as indicated. Appropriate concentrations of IPTG (10 μM for MT983; 50 μM for MT1315; no IPTG (leaky expression) for MT1200, MT1266, and MT1288) were added to the plates. Plates were scanned after incubation at 37°C for 6 days (6D) or for 14 days (14D), as indicated. Glyce glycerol, Pyru pyruvate.

Fig 6. Growth of Δrne ydfV::Tn10 Δrng E. coli strains complemented with various RNases.

Cultures of MT658 (a), MT1113 (b), MT1167 (c), MT1285 (d), MT1136 (e), MT1137 (f), or MT1140 (g) were spread onto M9-pyruvate plates (0.6% gellan gum) containing (0.1% ara) or lacking [ara(-)] 0.1% L-(+)-arabinose, as indicated. Appropriate concentrations of IPTG (10 μM for MT1136, MT1167, and MT1285; 50 μM for MT1140; no IPTG (leaky expression) for MT658, MT1113, and MT1137) were added to the plates. Plates were scanned after incubation at 37°C for 6 days (6D) or 14 days (14D), as indicated. A summary of the results (combined with the results in Fig 2) is shown on the right-hand side.