Role of the cold-box region in the 5' untranslated region of the cspA mRNA in its transient expression at low temperature in Escherichia coli

Abstract

Upon temperature downshift, a group of proteins called cold shock proteins, such as CspA, CspB, and CsdA, are transiently induced in Escherichia coli. However, when the 5' untranslated region (5' UTR) of cspA mRNA is overproduced at low temperature, the expression of cold shock genes is prolonged or derepressed. It has been proposed that this effect is due to highly conserved 11-base sequences designated the "cold box" existing in the 5' UTRs of cspA, cspB, and csdA. Here, we demonstrate that the overproduction of the 5' UTR of not only cspA but also cspB and csdA mRNAs causes derepression of all three genes at the same time. Conversely, when the cold-box region was deleted from the cspA 5' UTR its derepression function was abolished. The amount of mRNA from the chromosomal cspA gene was much higher in cells overproducing the wild-type 5' UTR by means of a plasmid than it was in cells overproducing the cold-box-deleted 5' UTR. The stability of the chromosomal cspA mRNA in cells overproducing the wild-type 5' UTR was almost identical to that in cells overproducing the cold-box-deleted 5' UTR. Therefore, the derepression of cspA caused by overproduction of 5' UTR at the end of the acclimation phase occurs at the level of transcription but not by mRNA stabilization, indicating that the cold-box region plays a negative role in cspA transcription in cold shock-adapted cells. The role of the cold-box region was further confirmed with a cspA mutant strain containing a cold-box-deleted cspA gene integrated into the chromosome, which showed a high level of constitutive production of CspA but not CspB during exponential growth at low temperature.

FIG. 1

Effects of overproduction of the 5′ UTRs of cspA and cspB mRNAs. Pulse-labeling experiments were carried out as described in Materials and Methods. (A) Cultures of cells harboring different plasmids (pRS414, pA-LacZ, and pB-LacZ) were shifted from 37 to 15°C at mid-log phase (80 Klett units). The time point (CS time, time after cold shock) of pulse-labeling is shown above each lane. The same amount of the culture (0.15 ml) was analyzed at each time point by SDS–17.5% polyacrylamide gel electrophoresis. The labeling times at 37 and 15°C were 5 and 15 min, respectively. The positions of CspA, CsdA, and β-galactosidase (β-gal) are indicated by arrows. Lanes 1 to 3, CL83 cells harboring the vector only; lanes 4 to 6, CL83 cells harboring pA-LacZ; lanes 7 to 9, CL83 cells harboring pB-LacZ. Molecular mass (in kilodaltons) is shown on the left. (B) Cells with pA-LacZ 3 h after cold shock (panel A, lane 6) were analyzed by two-dimensional gel electrophoresis. Only a portion of the autoradiogram is shown. Positions of CspA, CspB, and CspG are indicated by arrows. (C) Cells with pB-LacZ 3 h after cold shock (panel A, lane 9) were analyzed as described for panel B.

FIG. 2

Effects of overproduction of the 5′ UTR of csdA mRNA. Cultures of cells harboring the indicated plasmids were shifted from 37 to 15°C at mid-log phase (80 Klett units) and incubated at 15°C for 3 h. Cells were then labeled with [³⁵S]methionine for 15 min as described in Materials and Methods. The same amount of the culture (0.15 ml) was used for each labeling experiment and analyzed by SDS–17.5% polyacrylamide gel electrophoresis. Lane 1, CL83 cells harboring the vector pUC19; lane 2, CL83 cells harboring pUC19-600 for overproduction of the 5′ UTR of cspA mRNA; lane 3, CL83 cells harboring plmcsdA for overproduction of the 5′ UTR of csdA mRNA. Positions of CspA and CsdA are indicated by arrows. Molecular mass markers (in kilodaltons) are shown on the left.

FIG. 3

Effects of deletion of the cold-box region. Pulse-labeling experiments were carried out as described in Materials and Methods. Cells harboring p6mTEK, p2JTEK, and pΔ24T were shifted from 37 to 15°C at mid-log phase (80 Klett units) and then labeled with [³⁵S]methionine for 15 min 3 h after cold shock. The plasmids were used to overproduce cspA mRNA as follows: p6mTEK from bases +1 to +6, p2JTEK from bases +1 to +25, and pΔ24T from bases +1 to +143 with a deletion of the cold-box region from +3 to +24. Protein expression patterns were analyzed by SDS–17.5% polyacrylamide gel electrophoresis. Lane 1, CL83 cells harboring p6mTEK; lane 2, CL83 cells harboring p2JTEK; lane 3, CL83 cells harboring pΔ24T. The positions of CspA and CsdA are indicated by arrows. Molecular mass markers (in kilodaltons) are shown on the left.

FIG. 4

Detection of cspA transcripts in cells overproducing the 5′ UTR of cspA mRNA (from bases +1 to +143) with and without the cold-box region from bases +3 to +24. Total RNA was isolated from CL83 cells carrying pUC19-600 (control; lanes 1 and 2) and pΔ24T (cold-box deletion; lanes 3 and 4) after 3 h of incubation at 15°C. Primer extension experiments were carried out as described in Materials and Methods. Two primers were used in order to detect the cspA transcript from the chromosomal cspA gene (primer 3551; lanes 1 and 3) and the transcripts from both plasmids and the chromosome (primer 4592; lanes 2 and 4). The products of primer extension were separated by 7 M urea–6% acrylamide gel electrophoresis. The positions of the products of the different primers are indicated by arrows. CS: 3 hr, 3 h after cold shock.

FIG. 5

Stability of chromosomal cspA transcript in cells overproducing cspA 5′ UTR. CL83 cells harboring pUC19-600 for the 5′ UTR of the wild-type cspA gene and pΔ24T for the cold-box-deleted 5′ UTR of cspA were grown to mid-log phase and then shifted to 15°C for 3 h. Rifampin (200 μg/ml) was added to the culture, and RNA was extracted at 0, 5, 10, 20, and 30 min after the addition of rifampin. Primer extension was carried out, and the densities of products were analyzed by a phosphorimager and plotted. The amounts of transcripts at the 0 time point were taken as 100.

FIG. 6

Constitutive high expression of cspA at 15°C resulted from the deletion of the cold-box region from the chromosomal cspA gene. Strain WB003 (ΔCB), containing the cspA gene with a 22-base deletion from bases +3 to +24 in the 5′ UTR, and its parent strain (wild type), JC7623, were used for labeling cellular proteins. Labeling was carried out at 0.5, 7, and 32 h after a temperature downshift from 37 to 15°C, as previously described (8), and labeled proteins were analyzed by two-dimensional gel electrophoresis. The entire gel is shown only for strain WB003 at 37°C (A). Cell cultures were diluted before they reached the stationary phase, and only the lower parts are shown for 0.5-, 7-, and 32-h time points at 15°C. The positions of CspA are indicated by arrowheads.