A Translation-Aborting Small Open Reading Frame in the Intergenic Region Promotes Translation of a Mg2+ Transporter in Salmonella Typhimurium

Abstract

Bacterial mRNAs often harbor upstream open reading frames (uORFs) in the 5' untranslated regions (UTRs). Translation of the uORF usually affects downstream gene expression at the levels of transcription and/or translation initiation. Unlike other uORFs mostly located in the 5' UTR, we discovered an 8-amino-acid ORF, designated mgtQ, in the intergenic region between the mgtC virulence gene and the mgtB Mg²⁺ transporter gene in the Salmonella mgtCBRU operon. Translation of mgtQ promotes downstream mgtB Mg²⁺ transporter expression at the level of translation by releasing the ribosome-binding sequence of the mgtB gene that is sequestered in a translation-inhibitory stem-loop structure. Interestingly, mgtQ Asp2 and Glu5 codons that induce ribosome destabilization are required for mgtQ-mediated mgtB translation. Moreover, the mgtQ Asp and Glu codons-mediated mgtB translation is counteracted by the ribosomal subunit L31 that stabilizes ribosome. Substitution of the Asp2 and Glu5 codons in mgtQ decreases MgtB Mg²⁺ transporter production and thus attenuates Salmonella virulence in mice, likely by limiting Mg²⁺ acquisition during infection.IMPORTANCE Translation initiation regions in mRNAs that include the ribosome-binding site (RBS) and the start codon are often sequestered within a secondary structure. Therefore, to initiate protein synthesis, the mRNA secondary structure must be unfolded to allow the RBS to be accessible to the ribosome. Such unfolding can be achieved by various mechanisms that include translation of a small upstream open reading frame (uORF). In the intracellular pathogen Salmonella enterica serovar Typhimurium, translation of the Mg²⁺ transporter mgtB gene is enhanced by an 8-amino-acid upstream ORF, namely, mgtQ, that harbors Asp and Glu codons, which are likely to destabilize ribosome during translation. Translation of the mgtQ ORF promotes the formation of a stem-loop mRNA structure sequestering anti-RBS and thus releases the mgtB RBS. Because mgtQ-mediated MgtB Mg²⁺ transporter production is required for Salmonella virulence, this pathogen seems to control the virulence determinant production exquisitely via this uORF during infection.

Keywords: intergenic region; ribosome destabilization; translation-inhibitory stem-loop structure; uORF.

FIG 1

Regulation of the Salmonella mgtCBRU virulence operon by the mgtQ short ORF. (A) The phosphorylated PhoP response regulator binds to the mgtCBRU promoter and initiates transcription. Two short ORFs in the 5′ leader region, mgtM and mgtP, control transcription elongation in response to intracellular ATP and charged tRNA^Pro levels via transcription attenuation-like mechanisms. The 8-aa mgtQ ORF is located in the 219-nt intergenic region between mgtC and mgtB genes and harbors Asp and Glu codons at positions 2 and 5. The sequence overlapping the mgtQ ORF has the potential to adopt alternative RNA secondary structures (stem-loops 1:2 and 3:4 versus 2:3) and the mgtB RBS is occluded within the stem-loop 3:4 structure. The Asp and Glu codons at mgtQ appear to induce ribosome destabilization and promote the formation of stem-loop 2:3, thus releasing the RBS of the mgtB gene and enhancing mgtB translation. Positions of nucleotide substitutions used in the experiments presented in Fig. 4 are indicated. (B) Alignment of the deduced amino acid sequences of the mgtQ orthologs in the mgtC-mgtB intergenic regions from Salmonella enterica, Erwinia tasmaniensis, Yersinia pestis, Yersinia enterocolitica, and Serratia proteamaculans. Sequences in red correspond to Pro codons, and sequences in green correspond to Asp and Glu codons. Asterisks correspond to positions conserved in all listed species.

FIG 2

The mgtQ ORF is translated in vivo. (A) Schematic representation of mgtQ′-gfp constructs. (B) Fluorescence produced by wild-type Salmonella (14028s) harboring the plasmid vector (ptGFP), or derivatives with a gfp translational fusion to the last mgtQ codon (pmgtQ′-gfp), or following the stop mgtQ codon (pmgtQ _STOP′-gfp). Bacteria were grown for 4 h in N-minimal medium containing 10 mM Mg²⁺ as described in Materials and Methods. The means and standard deviations (SD) from three independent measurements are shown.

FIG 3

mgtQ translation affects mgtB expression at a translational level but not transcriptional level. (A) Schematic representation of mgtB-gfp constructs used in this experiment. (B and C) Fluorescence produced by wild-type Salmonella (14028s) harboring a plasmid with either a gfp gene translationally (B) or transcriptionally (C) fused to the mgtB gene with the wild-type mgtQ (mgtQ_WT) or an mgtQ mutant with the start codon substituted by the TAG stop codon (mgtQ_ATG→TAG). Bacteria were grown for 5 h in N-minimal medium containing 0.01 mM (inducing) or 10 mM (noninducing) Mg²⁺ as described in Materials and Methods. The means and SD from three independent measurements are shown.

FIG 4

Formation of alternative stem-loop structures that include mgtQ affects downstream mgtB translation. (A) Schematic representation of a model showing predicted alternative stem-loop structures (1:2 and 3:4 versus 2:3) that are associated with the mgtQ ORF. (B) Fluorescence produced by wild-type Salmonella (14028s) harboring the plasmid vector (vector), or derivatives with a gfp translational fusion to the mgtB gene that includes the upstream intergenic region (wild-type), substitution mutations in the stem regions 1, 2, 3, or 4 that hinder stem-loop formation (1:2, 2:3, and/or 3:4), or compensatory mutations that recover the formation of stem-loops 1:2, 2:3, or 3:4. A strain harboring a plasmid with a gfp translational fusion derived from the p_lac1-6 promoter was used as a positive control. Bacteria were grown for 4 h in N-minimal media containing 0.01 mM Mg²⁺ as described in Materials and Methods. The means and SD from three independent measurements are shown. (C) Fluorescence produced by wild-type Salmonella (14028s) harboring the plasmid vector (vector), or derivatives with a gfp transcriptional fusion to the mgtB gene that includes the upstream intergenic region (wild-type), substitution mutations described in panel B. A strain harboring a plasmid with a gfp transcriptional fusion derived from the p_lac1-6 promoter was used as a positive control. Bacteria were grown for 4 h in N-minimal media containing 0.01 mM Mg²⁺ as described in Materials and Methods. The means and SD from three independent measurements are shown.

FIG 5

Stem-loop formation but not mgtQ Pro codon is required for mgtQ-mediated mgtB translation. (A and B) Fluorescence produced by wild-type Salmonella (14028s) harboring the plasmid vector (vector), or derivatives with a gfp translational (A) or transcriptional (B) fusion to the mgtB gene that includes the wild-type mgtQ, nucleotide substitution mutations of the conserved proline codon replaced by a synonymous mutation (CCG) or nonsynonymous mutations, including Gly (GGG), Leu (CTC), His (CAC), and Arg (CGC). The strains harboring a plasmid with a gfp translational fusion derived from the p_lac1-6 promoter were used as positive controls. Bacteria were grown for 5 h in N-minimal media containing 0.01 mM Mg²⁺ as described in Materials and Methods. The means and SD from three independent measurements are shown.

FIG 6

Asp2 and Glu5 codons in mgtQ are required for mgtB translation. (A) Schematic representation of mgtQ′-gfp constructs used in this experiment. (B) Schematic representation of mgtB′-gfp constructs used in this experiment. (C) Fluorescence produced by wild-type Salmonella (14028s) by rpmE1 (EN1119), rpmE2 (EN1120), or rpmE1 rpmE2 (EN1368) Salmonella isolates harboring the promoterless plasmid vector (ptGFP), or by derivatives with gfp translational fusions to the wild-type mgtQ (pmgtQ′-gfp) or Asp2, Glu5 to Ala-substituted mgtQ (pmgtQ_2,5Ala′-gfp). (D) Fluorescence produced by wild-type Salmonella (14028s), by rpmE1 (EN1119), rpmE2 (EN1120), or rpmE1 rpmE2 double mutant (EN1368) Salmonella harboring the plasmid vector (vector), or by Salmonella derivatives with a gfp translational fusion to the mgtB gene that includes the wild-type mgtQ or nucleotide substitution mutation of the Asp2 and Glu5 codons replaced with Ala. Bacteria were grown for 4 h in N-minimal media containing 0.01 mM Mg²⁺ as described in Materials and Methods. The means and SD from three independent measurements are shown.

FIG 7

mgtQ translation promotes MgtB production and is required for Salmonella virulence in mice. (A and B) Western blot analysis of crude extracts prepared from wild-type, rpmE1, rpmE2, or rpmE1 rpmE2 Salmonella strains with either the wild-type mgtQ (14028s, EN1119, EN1120, or EN1368, respectively) or mgtQ_2,5Ala (EN1389, EN1408, EN1409, or EN1414, respectively) gene probed with anti-MgtB (A) or anti-Fur (B) antibodies to detect MgtB or Fur proteins, respectively. Bacteria were grown for 5 h in N-minimal media containing 0.01 mM Mg²⁺ as described in Materials and Methods. (C) The mgtQ_2,5Ala substitution attenuates Salmonella virulence in mice. Survival of C3H/HeN mice inoculated intraperitoneally with approximately 3000 CFU of wild-type (14028s), mgtQ_2,5Ala mutant (EN1389), and mgtB deletion mutant (EN481) Salmonella strains.