H-NS represses inv transcription in Yersinia enterocolitica through competition with RovA and interaction with YmoA

Abstract

Yersinia enterocolitica is able to efficiently invade Peyer's patches with the aid of invasin, an outer member protein involved in the attachment and invasion of M cells. Invasin is encoded by inv, which is positively regulated by RovA in both Y. enterocolitica and Yersinia pseudotuberculosis while negatively regulated by YmoA in Y. enterocolitica and H-NS in Y. pseudotuberculosis. In this study we present data indicating H-NS and RovA bind directly and specifically to the inv promoter of Y. enterocolitica. We also show that RovA and H-NS from Y. enterocolitica bind to a similar region of the inv promoter and suggest they compete for binding sites. This is similar to recently published data from Y. pseudotuberculosis, revealing a potentially conserved mechanism of inv regulation between Y. enterocolitica and Y. pseudotuberculosis. Furthermore, we present data suggesting H-NS and YmoA form a repression complex on the inv promoter, with H-NS providing the binding specificity and YmoA interacting with H-NS to form a repression complex. We also demonstrate that deletion of the predicted H-NS binding region relieves the requirement for RovA-dependent transcription of the inv promoter, consistent with RovA acting as a derepressor of H-NS-mediated repression. Levels of H-NS and YmoA are similar between 26 degrees C and 37 degrees C, suggesting that the H-NS/YmoA repression complex is present at both temperatures, while the levels of rovA transcript are low at 37 degrees C and high at 26 degrees C, leading to expression of inv at 26 degrees C. Expression of RovA at 37 degrees C results in transcription of inv and production of invasin. Data presented here support a model of inv regulation where the level of RovA within the cell governs inv expression. As RovA levels increase, RovA can successfully compete for binding to the inv promoter with the H-NS/YmoA complex, resulting in derepression of inv transcription.

FIG. 1.

Effect of hns on the expression of inv. (A) Cultures of MC4100 containing pinv-gfp and VM1303 (an hns mutant) containing pinv-gfp or pinv-gfp and phns were grown overnight in triplicate at 26°C. Fluorescence was calculated by dividing average fluorescence by the OD₆₀₀. WT refers to MC4100 containing pinv-gfp, hns⁻ refers to VM1303 containing pinv-gfp, and hns⁻/phns refers to VM1303 containing pinv-gfp and phns. (B) Cultures of JB41v containing the plasmids phns, pymoA (pEll21), or pWKS30strspec were grown overnight in triplicate at 26°C and assayed for expression of inv-phoA. WT refers to JB41v containing pWKS30strspec as a vector control.

FIG. 2.

Ability of RovA, H-NS, and YmoA to interact with inv. Randomly ³²P-labeled inv (−344 to +146) was incubated with purified RovA, H-NS, or YmoA at room temperature for 15 min and run on a 3.5% or 5% nondenaturing polyacrylamide gel. (A) RovA; (B) H-NS. In panels A and B, 1 μg of salmon sperm was included in all of the reaction mixtures. (C) YmoA. The + indicates the presence of 1 μg of salmon sperm; − indicates the absence of salmon sperm. The concentration of protein is shown across the top of the gel (in μM).

FIG. 3.

Alignment of inv promoters. Shown is an alignment of the RovA and H-NS binding region of inv from Y. enterocolitica and Y. pseudotuberculosis. Conserved bases are shaded in black, RovA binding sites are underlined in black, and H-NS bindings sites are indicated by a gray underline. The RovA and H-NS binding sites are based on DNase I protection data from Y. pseudotuberculosis (21). The start of transcription, marked +1, was previously determined (41, 46).

FIG. 4.

Competition between RovA and H-NS for the inv promoter. (A) Randomly ³²P-labeled inv (−194 to −34) was incubated for 5 min with purified RovA (0.5 μM), shown across the top of the gel. To these reaction mixtures increasing concentrations of H-NS were added, as shown across the top of the gel (in μM), and incubated for 10 min. Samples were run on a 5% nondenaturing polyacrylamide gel. (B) Randomly ³²P-labeled inv (−194 to −34) was incubated for 5 min with purified H-NS (0.6 μM), shown across the top of the gel. To these reaction mixtures increasing concentrations of RovA were added, as shown across the top of the gel (in μM), and incubated for 10 min. Samples were run on a 5% nondenaturing polyacrylamide gel.

FIG. 5.

Levels of hns in YVM567c. JB41c (WT) and YVM567c (ymoB::Tn10) were grown overnight at 37°C, and cultures were then subcultured to an OD₆₀₀ of 0.2 and grown to an OD₆₀₀ of 2.0. Transcriptional levels of hns were determined by Northern blotting. Levels of H-NS in the two strains are shown below the graph from samples grown in the same manner as for the Northern blot assay. Equal OD equivalents were loaded for each strain for the Western analysis, and data shown are from the same gel.

FIG. 6.

Ability of H-NS and YmoA to form a repression complex on the inv promoter. (A) Randomly ³²P-labeled inv (−344 to +146) was incubated with purified H-NS and YmoA at room temperature for 15 min and run on a 5% nondenaturing polyacrylamide gel. (B) Randomly ³²P-labeled lacZ was incubated with purified H-NS and YmoA at room temperature for 15 min and run on a 5% nondenaturing polyacrylamide gel. The concentration of YmoA is shown across the top of the gel (in μM), and H-NS was added at a constant concentration of 0.1 μM. The open arrow indicates an H-NS-inv complex, with the closed arrow indicating an H-NS/YmoA-inv complex.

FIG. 7.

Effect of promoter truncations on the expression of inv-gfp in WT and ΔrovA strains. (A) Schematic of the inv promoter with the predicted −35, −10, and +1 sites, along with the start codon (+107) and fusion junction (+170). The predicted RovA binding sites (black lines) from Y. pseudotuberculosis are shown above inv16, and the predicted H-NS binding sites (gray lines) are shown below (21). (B) JB580 (WT) and YVM927 (ΔrovA) strains were transformed with the inv-gfp promoter truncations and grown overnight at 26°C. Fluorescence was calculated by dividing the average fluorescence by OD₆₀₀. V indicates the pROBE-gfp[LVA], containing no promoter, and the numbers below the bars represent the different inv-gfp truncations shown in panel A. Shown below the numbers are the strains in which the truncations were tested.

FIG. 8.

Expression levels of inv, rovA, ymoA, and hns at early, middle, and late log phases of growth. (A) inv; (B) rovA; (C) hns; (D) ymoA. Cultures of WT bacteria were grown overnight and subcultured to an OD₆₀₀ of 0.2. Approximately 4 OD₆₀₀ of cells were taken at early log (0.5 to 1.0 OD₆₀₀), middle log (1.1 to 2.5), and late log (2.6 to 3.5) phases of growth, and the RNA was extracted. Three biological replicates were performed, but because of high signal variability between replicates, representative graphs are shown for inv, rovA, ymoA, and hns. The trends shown here were the same for all of the replicates, and the expression levels of hns and ymoA between 26°C and 37°C never differed by more than twofold. Corresponding levels of H-NS and YmoA, determined by Western analysis as described in Materials and Methods, are indicated below their graphs. For both H-NS and YmoA, the inserts were taken from the same gel and the same exposure time.

FIG. 9.

Expression of inv at 37°C by induction of rovA. Cultures were grown overnight at 26°C or 37°C in triplicate and assayed for inv-lacZ expression and relative levels of RovA, which are shown below the graph. WT and ΔrovA strains are shown on the ends of the graph, and the ΔrovA+pBADrovA mutant is shown in the middle; increasing concentrations of arabinose (0.000002, 0.00002, 0.0002, and 0.002%) are indicated by the black triangle.