Complementation analysis of the cold-sensitive phenotype of the Escherichia coli csdA deletion strain

Abstract

The cold shock response of Escherichia coli is elicited by downshift of temperature from 37 degrees C to 15 degrees C and is characterized by induction of several cold shock proteins, including CsdA, during the acclimation phase. CsdA, a DEAD-box protein, has been proposed to participate in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. It is not clear which of the functions of CsdA play a role in its essential cold shock function or whether all do, and so far no protein has been shown to complement its function in vivo. Our screening of an E. coli genomic library for an in vivo counterpart of CsdA that can compensate for its absence at low temperature revealed only one protein, RhlE, another DEAD-box RNA helicase. We also observed that although not detected in our genetic screening, two cold shock-inducible proteins, namely, CspA, an RNA chaperone, and RNase R, an exonuclease, can also complement the cold shock function of CsdA. Interestingly, the absence of CsdA and RNase R leads to increased sensitivity of the cells to even moderate temperature downshifts. The correlation between the helicase activity of CsdA and the stability of mRNAs of cold-inducible genes was shown using cspA mRNA, which was significantly stabilized in the DeltacsdA cells, an effect counteracted by overexpression of wild-type CsdA or RNase R but not by that of the helicase-deficient mutant of CsdA. These results suggest that the primary role of CsdA in cold acclimation of cells is in mRNA decay and that its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature.

FIG. 1.

RhlE can complement the cold-sensitive phenotype of ΔcsdA cells at 15°C. E. coli wild-type cells were transformed with pACYC-duet plasmid as a control, and ΔcsdA cells were transformed with pACYC-duet plasmid alone as a control or containing csdA or rhlE. The cells were streaked on LB plates containing chloramphenicol (50 μg ml⁻¹) and incubated at 37°C and 15°C. The results obtained with the plates incubated at 37°C for 24 h and at 15°C for 72 h are presented.

FIG. 2.

Analysis of the role of acidic amino acids of the DEAD box in the helicase activity of CsdA. (A) E. coli wild-type cells were transformed with pACYC-duet plasmid as a control, and ΔcsdA cells were transformed with pACYC-duet plasmid alone as a control or expressing entire CsdA or C-terminal-truncated CsdA (CsdA_trn) carrying DEAD-box amino acid mutation D156A, E157A, or D159A; the corresponding mutant proteins are designated CsdA(AEAD), CsdA(DAAD) and CsdA(DEAA), respectively. The cells were streaked on LB plates containing chloramphenicol (50 μg ml⁻¹) and incubated at 37°C and 15°C. The results of the plate incubation at 37°C for 24 h and at 15°C for 72 h are presented. (B) Growth curves of wild-type cells with pACYC-duet vector alone, ΔcsdA cells, and ΔcsdA cells expressing wild-type CsdA (ΔcsdA+CsdA) or C-terminally truncated CsdA (ΔcsdA+CsdAtrn) grown at 15°C.

FIG. 3.

(A) Complementation of the cold-sensitive phenotype of the ΔcsdA cells by CspA and RNase R. E. coli wild-type cells were transformed with pACYC-duet plasmid as a control, and ΔcsdA cells were transformed with pACYC-duet plasmid alone as a control or expressing CsdA, CspA, or RNase R. The cells were streaked on LB plates containing chloramphenicol (50 μg ml⁻¹) and incubated at 37°C and 15°C. The results for the plates incubated at 37°C for 24 h and at 15°C for 72 h are presented. (B) DEAD-box RNA helicase: RhlB does not complement the cold-sensitive phenotype of the ΔcsdA cells. E. coli wild-type cells were transformed with pACYC-duet plasmid as a control, and ΔcsdA cells were transformed with pACYC-duet plasmid alone as a control or expressing CsdA or RhlB. The cells were streaked on LB plates containing chloramphenicol (50 μg ml⁻¹) and incubated at 37°C and 15°C. The results for the plates incubated at 37°C for 24 h and at 15°C for 120 h are presented.

FIG. 4.

Double-deletion strain of csdA and rnr has increased temperature sensitivity. E. coli wild-type and ΔcsdA, Δrnr, and ΔcsdAΔrnr cells were streaked on LB plates and incubated at 37°C, 30°C, and 20°C for the time intervals indicated (A), and E. coli wild-type, ΔcsdA, ΔrhlE, Δrnr, ΔcsdAΔrhlE, ΔcsdAΔrnr, ΔrhlEΔrnr, and ΔcsdAΔrhlEΔrnr cells were streaked on LB plates and incubated at 20°C for 48 h (B).

FIG. 5.

CsdA and RNase R, but not RNase II or PNPase, can complement the cold-sensitive phenotype of the ΔcsdAΔrnr cells. E. coli wild-type, ΔcsdA, Δrnr, and ΔcsdAΔrnr cells were transformed with pINIII plasmid as a control, and ΔcsdAΔrnr cells were transformed with pINIII plasmid alone as a control or with pINII-csdA, pINIII-rnr, pINIII-rnb, or pINIII-pnp expressing CsdA, RNase R, RNase II, or PNPase, respectively. The cells were streaked on LB plates containing ampicillin (50 mg ml⁻¹). The plates were incubated at 37°C, 20°C, and 15°C for the time intervals indicated.

FIG. 6.

Helicase activity of CsdA is essential for its role in mRNA degradation at low temperature. Cells from which total RNAs were isolated by the hot-phenol method are indicated above the respective lanes. The exponentially growing cells were cold shocked at 15°C for 1 h, and transcription was stopped by adding rifampin. Equal amounts of RNA samples were used for primer extension (0 min and 40 min time points for each type of cells) with deoxyoligonucleotide corresponding to the cspA gene.