PMC

lcrH and yopD regulate yopQ expression at a posttranscriptional step. Expression of yopQ was measured by fusing the reading frame of npt to either the yopQ promoter (pDA330), the promoter and 5" UTR (pDA183), or the 3"end of the yopQ reading frame specifying a translational YopQ-Npt fusion (pDA243). Plasmids were transformed into Y. enterocolitica W22703 (wild type) or the isogenic Δ(lcrH) (CT133) and Δ(yopD) (VTL2) mutant strains. Yersinia strains were grown in the presence or absence of calcium, and bacterial extracts were prepared by TCA precipitation. The concentration of Npt reporter was determined by immunoblotting and is reported as the ratio between mutant and wild-type cells. Data were averaged from more than three independent experiments. The standard deviation (±) is indicated.

GST-LcrH binds to YopD or YopB. Plasmid pDA326 was transformed into the Δ(lcrH) mutant Yersinia strain CT133, and the expression of GST-LcrH was induced by the addition of IPTG. Bacterial lysates were cleared by centrifugation, and GST-LcrH was purified by affinity chromatography on glutathione-Sepharose. (A) Purification was assessed on Coomassie-stained SDS-PAGE and by immunoblotting of load and eluate fractions. (B) When expressed in Δ(yopD) cells, subjected to affinity chromatography, and measured on Coomassie-stained SDS-PAGE, GST-YopD bound to LcrH but not to YopB (VTL2 harboring pDA255). Molecular size markers (lane M, in kilodaltons), lysate (L), and eluate fractions (1, 2, and 3) are indicated. (C) Purification of GST-YopB, GST-YopD, and GST-LcrH was measured by subjecting affinity chromatography load (lysate) and eluate fractions to immunoblotting. Chemiluminescent signals were scanned and quantified and are reported as the ratio of signal intensity between eluate and lysate. This experiment was performed in duplicate, with yersiniae grown in the presence and absence of calcium.

yopD mutation suppresses the signaling defect of the YopQ₋₁-Npt mutation. The secretion signal of yopQ, i.e., codons 1 to 15 fused to npt, was mutated by deleting a nucleotide immediately following the AUG translational start. The reading frameshift was corrected at the fusion site with the npt reporter, generating the YopQ₋₁-Npt fusion (pDA228). Wild-type Y. enterocolitica W22703 (wt), the Δ(yopN) mutant strain VTL1, and the Δ(yopD) mutant VTL2 were transformed with pDA228. Cultures were induced for type III secretion by growing yersiniae at 37°C in the absence of calcium. Cultures were centrifuged, and the extracellular medium was separated into supernatant (S) and bacterial pellet (P). The expression and location of YopQ₋₁-Npt and cytoplasmic chloramphenicol acetyltransferase (Cat) was measured by immunoblotting with specific antibody. The secretion of YopQ₋₁-Npt was quantified with an alpha imager: W22703, 0%; Δ(yopD) mutant VTL2, 58%; and the Δ(yopN) mutant strain VTL1, 0%. The relative concentration of the protein [YopQ₋₁-Npt] is indicated for each strain.

YopD is required for degradation of yopQ mRNA. (A) Plasmid pDA209 carries the cat gene and a transcriptional yopQ-npt fusion. The drawing displays the annealing positions of oligonucleotide primers (arrowheads). (B) Y. enterocolitica strains MC3 [Δ(yopQ)] and DA1 [Δ(yopDQ)] were transformed with pDA209 () and grown in either the presence or absence of calcium. RNA was purified and cDNA was synthesized (+ and − indicate the addition or omission of AMV reverse transcriptase, respectively) using oligonucleotides that anneal at the 3" end of yopQ-npt or cat. cDNA template was used for PCR amplification (arrowheads), and products were analyzed on ethidium bromide-stained agarose gel. The 1-kb DNA ladder was used for size calibration (lane M). (C) The ratio of npt/yopQ to npt transcripts in various strains and growth conditions was determined by quantifying fluorescent signals. (D) Cell extracts of Y. enterocolitica MC3(pDA209) and DA1(pDA209) were analyzed by immunoblotting with α-YopQ and α-Npt.

Synthesis and type III secretion of YopQ in wild-type and mutant yersiniae. (A) Y. enterocolitica W22703 (wild type) and isogenic mutant strains VTL1 [Δ(yopN)], VTL2 [Δ(yopD)], and KUM1 [Δ(yscV)] were grown in the presence and absence of calcium (low calcium is an inducing condition for type III secretion). Cultures were centrifuged, and proteins secreted into the culture medium (S, supernatant) were separated from the cell sediment (P, pellet). YopQ secretion was measured by immunoblotting with specific antiserum. (B) Y. enterocolitica strains W22703 (wild type), VTL2 [Δ(yopD)], and CT133 [Δ(lcrH)] were grown at 37°C in the presence and absence of calcium. Culture supernatants (S) and bacterial extracts (P) were separated on SDS-PAGE and analyzed by immunoblotting with specific antiserum (α-YopQ). Yersinia strains were transformed with plasmids encoding wild-type yopD (pVL41) (), gst-yopD (pDA255) (), lcrH (pDA325), or gst-lcrH (pDA326). (C) YopQ secretion in Y. enterocolitica strains carrying knockout mutations in both the yopD and ysc genes.

YopD and LcrH complexes bind yopQ mRNA. A transcriptional yopD-lcrH fusion (A), lcrH (C), or yopD (E) was cloned into pET16 (Novagen) and transformed into E. coli BL21(DE3). Protein was expressed by IPTG induction of T7 polymerase. Total cell extracts (T) were centrifuged, and the cleared supernatant (L, load) was applied to affinity chromatography on Ni-NTA. Flowthrough (F), column wash samples with increasing stringency (imidazole), and elution with 0.5 M imidazole were analyzed by separating proteins on SDS-15% PAGE and staining with Coomassie blue. The migration of molecular size markers is indicated. ³²P-labeled yopQ mRNA was obtained by in vitro transcription of pDA340 with [³²P]UTP and gel purification. ³²P-labeled yopQ mRNA (10 fmol) was incubated in the presence of increasing amounts of purified YopD and LcrH (0, 0.9, 1.8, 2.7, 3.6, 4.5, 5.4, 6.3, 7.2, 8.1, 9, and 27 ng) (B) or LcrH alone (D) and separated by electrophoresis on a 4% polyacrylamide gel. Specificity of yopQ mRNA binding (9 ng of YopD and LcrH to 10 fmol of ³²P-labeled yopQ mRNA) was assessed by adding excess unlabeled yopQ or E. coli tRNA (0, 10, 20, 50, 80, 100, and 500 fmol) (F).