Localization of Components of the RNA-Degrading Machine in Bacillus subtilis

Abstract

In bacteria, the control of mRNA stability is crucial to allow rapid adaptation to changing conditions. In most bacteria, RNA degradation is catalyzed by the RNA degradosome, a protein complex composed of endo- and exoribonucleases, RNA helicases, and accessory proteins. In the Gram-positive model organism Bacillus subtilis, the existence of a RNA degradosome assembled around the membrane-bound endoribonuclease RNase Y has been proposed. Here, we have studied the intracellular localization of the protein that have been implicated in the potential B. subtilis RNA degradosome, i.e., polynucleotide phosphorylase, the exoribonucleases J1 and J2, the DEAD-box RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase. Our data suggests that the bulk of these enzymes is located in the cytoplasm. The RNases J1 and J2 as well as the RNA helicase CshA were mainly localized in the peripheral regions of the cell where also the bulk of messenger RNA is localized. We were able to demonstrate active exclusion of these proteins from the transcribing nucleoid. Taken together, our findings suggest that the interactions of the enzymes involved in RNA degradation in B. subtilis are rather transient.

Keywords: RNA degradation; RNA degradosome; RNase Y; microbial cell biology; protein localization.

FIGURE 1

The strains harboring the GFP fusions to PnpA and RNase J1 show a wild type phenotype. Light microscopy of strains (A) 168 (WT), GP1698 (pnpA-gfp), and GP1748 (ΔpnpA) and (B) 168 (WT), GP1722 (rnjA-gfp), and GP2502 (ΔrnjA). The strains were grown in LB medium at 37°C to stationary phase. Both strains harboring the GFP fusions form short cells comparable to the wild type strain and are not elongated as their respective deletion mutants. Scale bar, 5 μm. WT, wild type.

FIGURE 2

RNases J1 and J2 form a complex in vivo. Bacillus subtilis GP1048 was cultivated in LB medium at 37°C to OD₆₀₀ of 1.0. Cells were disrupted and Strep-RNase J1 was purified by a StrepTactin column. CE, cell extract; W, wash fraction; E2, elution fraction 2. The identity of RNases J1 and J2 was proven by Western blot analysis (data not shown). The faint eluted bands at the top and the bottom of the gel correspond to PycA and AccB, respectively, the two biotin-containing proteins of B. subtilis (Meyer et al., 2011).

FIGURE 3

The strains harboring the GFP fusions to CshA, PfkA, and Enolase do not show growth defects. (A) Strains 168 (WT), GP1721 (cshA-gfp), and GP1035 (ΔcshA) were streaked on LB agar and incubated at 28°C overnight. The strain harboring the cshA-gfp fusion in the genome is able to grow under these conditions while the deletion mutant has a growth defect. (B) Strains 168 (WT), GP1720 (pfkA-gfp), and GP1747 (ΔpfkA) were streaked on CE minimal medium plates with 0.5% glucose and incubated for 48 h at 37°C. The strain with the pfkA-gfp fusion is able to grow under these conditions, in contrast to the deletion mutant. (C) Strains 168 (WT), GP1700 (eno-gfp), and GP594 (Δeno) were streaked on LB plates and incubated at 37°C overnight. The strain harboring the eno-gfp fusion can grow under these conditions, while the deletion mutant is unable to grow.

FIGURE 4

Membrane localization of RNase Y. Fluorescence microscopy of the strain GP1684 (Pxyl-rny-gfp) grown in LB medium at 28°C to stationary phase. The expression of the fusion protein was induced by addition of 0.1% xylose to the culture medium. The membrane was stained with Nile Red. RNase Y-GFP co-localizes with the Nile Red staining, confirming the membrane localization of the protein. Scale bar, 2 μm. PC, phase contrast.

FIGURE 5

The RNA-degrading exoribonuclease PnpA, as well as the glycolytic enzymes enolase and PfkA are localized to the cytoplasm, where they appear uniformly distributed. Fluorescence microscopy of strains harboring GFP fusions. (A) The strains GP1698 (pnpA-gfp) was grown in LB medium at 28°C to mid-exponential phase. The GFP fusion protein can is localized to the cytoplasm, where it appears homogeneously distributed. (B) Strain GP1700 (eno-gfp) was grown to mid-exponential phase in LB medium at 28°C or 37°C. The protein appears widespread in the cytoplasm at both temperatures but at 37°C some cells show bright spots localized at the poles. (C) The strain GP1720 (pfkA-gfp) was grown in LB medium at 28°C to mid-exponential phase and the fusion protein is evenly distributed within the cytoplasm. Scale bar, 2 μm. PC, phase contrast.

FIGURE 6

Polar localization of the RNases J1 and J2 and the DEAD-box RNA helicase, CshA. Fluorescence microscopy of strains GP1694 (Pxyl-rnjA-gfp), GP1695 (Pxyl-rnjB-gfp), and GP1721 (cshA-gfp). The cells were grown in LB medium at 28°C to mid-exponential phase. The expression of RNase J1-GFP and RNase J2-GFP was induced by addition of 0.1% xylose to the culture medium. The nucleoid was stained by DAPI as described in the Material and Methods section. The proteins are distributed in the cytoplasm concentrating at the poles. The concentration of the proteins is lower at the center of each cell, where the nucleoid is positioned as can be observed by DAPI stain. Scale bar, 2 μm. PC, phase contrast.

FIGURE 7

The sub-polar localization of RNases J1 and J2 is lost upon inhibition of RNA synthesis by rifampicin. Fluorescence microscopy of strains (A) GP1694 (Pxyl-rnjA-gfp) and (B) GP1695 (Pxyl-rnjB-gfp). Bacteria were grown in LB medium at 37°C to mid-exponential phase, when rifampicin or methanol were added to the cultures as described above. The expression of RNase J1-GFP and RNase J2-GFP was induced by addition of 0.1% xylose to the culture medium. The nucleoid was stained by DAPI as described in the Material and Methods section. The sub-polar localization of both RNases J1 and J2 is lost as the RNA synthesis is inhibited and the proteins appear evenly distributed in the cytoplasm. Upon addition of rifampicin the nucleoid, stained by DAPI, spreads occupying the majority of the cell volume. Scale bar, 2 μm. PC, phase contrast.