Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3' ends

Abstract

Timely turnover of RNA is an important element in the control of bacterial gene expression, but relatively few specific targets of RNA turnover regulation are known. Deletion of the Bacillus subtilis pnpA gene, encoding the major 3' exonuclease turnover enzyme, polynucleotide phosphorylase (PNPase), was shown previously to cause a motility defect correlated with a reduced level of the 32-gene fla/che flagellar biosynthesis operon transcript.fla/che operon transcript abundance has been shown to be inhibited by an excess of the small regulatory protein, SlrA, and here we find that slrA mRNA accumulated in the pnpA-deletion mutant. Mutation of slrA was epistatic to mutation of pnpA for the motility-related phenotype. Further, Rho-dependent termination was required for PNPase turnover of slrA mRNA. When the slrA gene was provided with a Rho-independent transcription terminator, gene regulation was no longer PNPase-dependent. Thus we show that the slrA transcript is a direct target of PNPase and that regulation of RNA turnover is a major determinant of motility gene expression. The interplay of specific transcription termination and mRNA decay mechanisms suggests selection for fine-tuning of gene expression.

Figure 1.

Increased level of slrA mRNA in the absence of PNPase. (A) Read data from RNA-Seq analysis of the slrA gene in pnpA⁺ (solid line) and ΔpnpA (dashed line) strains. Genome coordinates on the X-axis; normalized reads on the Y-axis. Regions of the slrA transcription unit are indicated below the data. (B) Northern blot analysis of slrA mRNA in Bacillus subtilis 168 and 3610 backgrounds. Ten microgram of total RNA was fractionated on a 6% denaturing polyacrylamide gel. FL, full-length mRNA; DI, decay intermediate. Marker lane contained 5′-end-labeled fragments of a TaqI digest of plasmid pSE420 (51).

Figure 2.

slrA mutation is epistatic to pnpA mutation for cell chaining. (A) Phase-contrast microscopy of cell chaining for the indicated strains. pnpA⁺, wild-type 3610 strain; slrA⁺, additional copy of slrA gene; ΔpnpA, deletion of gene encoding PNPase; ΔslrA, deletion of gene encoding SlrA; ΔpnpA ΔslrA, deletion of genes encoding PNPase and SlrA. (B) Overnight growth of strains in the presence of increasing concentrations of tetracycline.

Figure 3.

IPTG-induced slrA expression. (A) Northern blot analysis of slrA mRNA transcribed from the P_spac promoter. Ten microgram of total RNA was fractionated on a 6% denaturing polyacrylamide gel. Migration of 5′-end-labeled fragments of TaqI-digested pSE420 indicated at left. (B) Northern blot analysis of slrA mRNA half-life. Above each lane is time (min) after rifampin addition. Measured half-lives (average of two experiments) of specific bands are indicated on the right. Bands 1–4 are large enough to include the full slrA CDS. (C) Phase-contrast microscopy of cell chaining with increasing IPTG concentrations.

Figure 4.

Rho-dependent termination of slrA transcription. (A) Sequence and predicted secondary structure (52) of 3′-proximal region of slrA 3′ UTR. Open arrow indicates location of major 3′ end at nt 301, mapped by 3′ RACE in the pnpA⁺ strain. Location of additional 3′ ends mapped in the ΔpnpA strain indicated by short arrows. Numbering is from downstream of the slrA CDS. (B) Northern blot analysis of slrA mRNA in rho⁺ and Δrho strains. Ten microgram of total RNA was fractionated on a 1.0% MOPS-formaldehye agarose gel. RT, read-through. Migration of unlabeled RNA size markers indicated at left. (C) slrA gene schematic and location of slrA deletion constructs. Scale below is in base-pairs. (D) Northern blot analysis of P_spac-driven slrA mRNA from wild-type slrA gene and from deletion constructs. Ten microgram of total RNA was fractionated on a 6% denaturing polyacrylamide gel. (E) Northern blot analysis of slrA mRNA in the presence and absence of RNase R. Ten microgram of total RNA was fractionated on a 6% denaturing polyacrylamide gel.

Figure 5.

slrA with Rho-independent terminator. (A) Predicted secondary structure of the ermC transcription terminator. (B) Northern blot analysis of slrA mRNA carrying the ermC transcription terminator (slrATT). Ten microgram of total RNA was fractionated on a 6% denaturing polyacrylamide gel. (C) Phase-contrast microscopy of slrATT strains with increasing IPTG concentrations.