yopD and lcrH are required for the function of YscM1 and YscM2. Y. enterocolitica strains were analyzed for type III secretion as described in the legend to Fig. 1. (A) Y. enterocolitica strains W22703 (wild type), VTL2 [Δ(yopD)], CT133 [Δ(lcrH)], and EC2 [Δ(yscM1 yscM2)] were analyzed by immunoblotting with antisera raised against purified YscM1, YscM2, YopD, LcrH, and YscD. The type III machinery component YscD is located within bacteria. (B) Y. enterocolitica W22703 (wild type) harboring pDA259 (encoding Gst), pEC345 (encoding YscM1), or pEC348 (encoding YscM2) were cultured in −Ca²⁺. Plasmid-borne genes were overexpressed (⁺⁺) by adding 1 mM IPTG when shifting the temperature of bacterial cultures to induce the type III virulon. (C) Yersinia strains VTL2 [Δ(yopD)] and CT133 [Δ(lcrH)] were transformed with pEC345 and pEC348 and analyzed by immunoblotting.

Transcription of yopQ mRNA is not blocked by YscM1 and YscM2 overexpression. (A) Y. enterocolitica W22703 (wild type) and strains harboring plasmids pEC345 (encoding YscM1) and pEC348 (encoding YscM2) were analyzed for type III secretion as described in the legend to Fig. 1. Each fraction was analyzed by immunoblotting with antisera raised against purified YopQ. (B and C) Total RNA was isolated from the bacterial sediment (pellet), and RT-PCR analysis of yopQ mRNA was performed (+) or reverse transcriptase was omitted from the reaction mixture (−) prior to PCR amplification. The 549-bp yopQ ORF was analyzed by 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized on a UV light table. The size of the amplicon was compared to a molecular size standard (1 kb). Virulence plasmid DNA (pYVe227) was used as a positive control for the PCR amplification step.

The 5′ UTR of yopQ mRNA is the target of Gst-YscM1- and Gst-YscM2-mediated repression. Y. enterocolitica strains MC3 [Δ(yopQ)], VTL2 [Δ(yopD)], and CT133 [Δ(lcrH)] harboring plasmid pDA183, pDA184, pDA208, or pDA330 were cultured in −Ca²⁺ and analyzed for synthesis of YopE and Npt. Y. enterocolitica strains were transformed with pEC260 (encoding Gst), pEC347 (encoding Gst-YscM1), or pEC350 (encoding Gst-YscM2). The expression of pEC260-, pEC347-, or pEC350-carried genes was induced by the addition of 1 mM IPTG when shifting the temperature of bacterial cultures to induce the type III virulon. Quantification of immunoreactive signals is reported as the percent amount of the Npt signal compared to the signal for bacteria overexpressing Gst.

The 5′ UTR of yopE mRNA is the target of Gst-YscM1- and Gst-YscM2-mediated repression. (A) Y. enterocolitica strain MC3 [Δ(yopQ)] harboring plasmid pEC80 or pEC102 was cultured in −Ca²⁺ and analyzed for synthesis of YopE and Npt. Y. enterocolitica strains were transformed with pEC260 (encoding Gst), pEC347 (encoding Gst-YscM1), or pEC350 (encoding Gst-YscM2). The expression of pEC260-, pEC347-, or pEC350-carried genes was induced by the addition of 1 mM IPTG when shifting the temperature of bacterial cultures to induce the type III virulon. Quantification of immunoreactive signals is reported as the percent amount of the Npt signal compared to the signal for bacteria overexpressing Gst. (B) A conserved nucleotide sequence was identified in the 5′ UTR of yopQ, yopE, yopH, yopM, yopP, yopT, yscM1, yscM2, and sycH transcripts. Conserved sequences of individual genetic loci with nucleotide positions (relative to the translational AUG start codon) are indicated in the box with a consensus sequence listed at the bottom. Small boxes represent predicted ribosome binding sites.

The 5′-AUAAA-3′ elements of the 5′ UTR of yopQ are sufficient to impose Gst-YscM1- and Gst-YscM2-mediated repression when fused to the 5′ UTR of npt. (A) Illustration of the truncation strategy of the 5′ yopQ UTR. Sequences highlighted in boldface are predicted to be necessary for yscM1- and yscM2-mediated regulation. Arrows indicate the inclusion of conserved sequence (bold arrow) with truncations in both the 5′ and 3′ directions. Each truncation was appended to the 5′ end of the 5′ npt UTR. (B) Y. enterocolitica strain MC3 [Δ(yopQ)] harboring plasmids pEC52 to pEC58 and pEC70 to pEC74 was cultured in −Ca²⁺ and analyzed for synthesis of Npt. Y. enterocolitica strains were transformed with pEC260 (encoding Gst), pEC347 (encoding Gst-YscM1), or pEC350 (encoding Gst-YscM2). The expression of pEC260-, pEC347-, or pEC350-carried genes was induced by the addition of 1 mM IPTG when shifting the temperature of bacterial cultures to induce the type III virulon. Quantification of immunoreactive signals is reported as the percent amount of the Npt signal compared to the signal for bacteria overexpressing Gst.

Regulation of yop expression in Y. enterocolitica. Y. enterocolitica strains were cultured in TSB supplemented with 5 mM CaCl₂ (+Ca²⁺) or 5 mM EGTA (−Ca²⁺) for 2 h at 26°C and then induced for type III secretion at 37°C for 3 h. Cultures were centrifuged to separate the bacterial pellet (P) from the culture supernatant (S). Proteins in both fractions were precipitated with TCA and analyzed by SDS-PAGE and immunoblotting. (A) Protein of Y. enterocolitica strain W22703 (wild type) was analyzed by immunoblotting for the synthesis and secretion of YopQ, YopE, and YopD. (B and C) Synthesis and secretion of YopQ in various Y. enterocolitica strains were analyzed by immunoblotting with antisera raised against purified YopQ. Plasmid-encoded genes were overexpressed (⁺⁺) by adding 1 mM IPTG when shifting the temperature of bacterial cultures to induce the type III virulon.

Nonsecretable YscM1 and YscM2 also require yopD and lcrH for function. Y. enterocolitica strains were analyzed for type III secretion as described in the legend to Fig. 1. Y. enterocolitica strains W22703 (wild type) (A and C) and VTL2 [Δ(yopD)] and CT133 [Δ(lcrH)] (B) were analyzed by immunoblotting with antisera raised against purified YopQ, Gst, YscM1, YscM2, and Cat. Plasmids pEC260 (encoding Gst), pEC347 (encoding Gst-YscM1), pEC350 (encoding Gst-YscM2), pDA325 (encoding LcrH), pKR12 (encoding LcrH and YopD), and pEC441 (encoding SycH) were transformed into Yersinia strains, and expression was induced by the addition of 1 mM IPTG when shifting the temperature of bacterial cultures to induce the type III virulon.

Gst-YscM1 and Gst-YscM2 block the expression of plasmid-encoded YopQ at a posttranscriptional step. (A) Y. enterocolitica MC3 [Δ(yopQ)] harboring plasmid pDA218, pDA209, or pDA243 was cultured in −Ca²⁺ and analyzed for synthesis and secretion of YopQ as described in the legend to Fig. 1 with YopQ- and Npt-specific antisera. (B) Y. enterocolitica MC3 [Δ(yopQ)] harboring pDA218 was transformed with pEC260 (encoding Gst), pEC347 (encoding Gst-YscM1), or pEC350 (encoding Gst-YscM2). The expression of pEC260-, pEC347-, or pEC350-carried genes was induced by the addition of 1 mM IPTG when shifting the temperature of bacterial cultures to induce the type III virulon. Protein precipitated from total cultures was analyzed by immunoblotting for the synthesis of YopQ, Gst, or Cat (plasmid encoded) and YopE, YopD, or LcrH (virulence plasmid encoded) with specific antisera. (C) Y. enterocolitica MC3 [Δ(yopQ)] harboring plasmid pDA209 or pDA243 was cultured in −Ca²⁺ and analyzed for synthesis of YopQ and Npt. Y. enterocolitica MC3 [Δ(yopQ)] harboring pDA209 or pDA243 was transformed with pEC260 (encoding Gst), pEC347 (encoding Gst-YscM1), or pEC350 (encoding Gst-YscM2). The expression of pEC260-, pEC347-, or pEC350-carried genes was induced as described for panel B. Quantification of immunoreactive signals is reported as the percent amount of the Npt signal compared to the signal for bacteria overexpressing Gst.

The yopQ promoter is not required for class II-mediated posttranscriptional regulation of yopQ. Y. enterocolitica strains W22703 (wild type) and EC6 [Δ(yopD lcrH yscM1 yscM2)] were transformed with pEC83 or pEC84 (A). Both plasmids encode an npt reporter gene under the control of the npt promoter and either with or without 5′ yopQ UTR sequences. Y. enterocolitica strains were cultured in TSB supplemented with 5 mM CaCl₂ (+Ca²⁺), and the type III virulon was induced at 37°C for 0, 60, and 120 min. Secretion was quenched by precipitating protein from total cultures followed by immunoblot analysis for the synthesis of Npt with specific antisera. Data generated in panel B were quantified and analyzed for panel C with chemiluminescent signals that were visualized on a Fluorchem 8800 Imaging System (Alpha Innotech). wt, wild type.

The sequence 5′-AAUAAAU-3′ in the 5′ UTR of yopE mRNA is necessary for Gst-YscM1- and Gst-YscM2-mediated repression. (A and B) Y. enterocolitica strain MC3 [Δ(yopQ)] harboring plasmids pEC112, pEC138 to pEC145, and pEC148 was cultured in −Ca²⁺ and analyzed for synthesis of Npt. Y. enterocolitica strains were transformed with pEC260 (encoding Gst), pEC347 (encoding Gst-YscM1), or pEC350 (encoding Gst-YscM2). The expression of pEC260-, pEC347-, or pEC350-carried genes was induced by the addition of 1 mM IPTG when shifting the temperature of bacterial cultures to induce the type III virulon. Quantification of immunoreactive signals is reported as the percent amount of the Npt signal compared to the signal for bacteria overexpressing Gst. Asterisks indicate nucleotide positions in each construct where transversion substitutions were introduced. Dashed lines indicate sites of positional introduction of conserved sequence in the 5′ npt UTR of the 5′ yopE UTR (pEC148) and the −28 5′ yopQ UTR (pEC112) with substituted nucleotides indicated in boldface italics. Wild-type 5′ yopE UTR (pEC80) and 5′ npt UTR (pDA330) are included for reference. The underlined sequence CAU in pEC80 represents insertion of nucleotides at the NdeI fusion site. Small boxes represent predicted ribosome (16S rRNA) binding sites (Shine-Dalgarno box). (C and D) Graphical representation of the data generated in panels A and B, respectively, with quantified data from pEC80 and pDA330 included for reference. All chemiluminescent signals were visualized, quantified, and analyzed on a Fluorchem 8800 Imaging System (Alpha Innotech).

Model for the regulation of yop genes by class II gene products. (A) Upon host entry and exposure to 37°C, glutamate (serum amino acid), and 1.2 mM calcium, YopD, LcrH, YscM1, and YscM2 prevent the translation of yop mRNAs by recognizing a feature in the 5′ UTR. (B) Albumin (serum protein) activates the type III secretion of YopD. (C) Insertion of type III needles into the plasma membrane of macrophages triggers a calcium signal that activates the type III targeting of YscM1 and YscM2 via binding to the SycH chaperone. Secretion of YopD and targeting of YscM1 and YscM2 relieve the inhibition of yop mRNA translation and activate Yersinia type III injection of effector Yops.