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°C to 15°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 ⌬csdA cells, an effect counteracted by overexpression of wild-type CsdA or RNase R but not by that of the helicasedeficient 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.

RNA remodeling and gene regulation by cold shock proteins

“…Again, this effect was counteracted by overexpression of the wild-type CsdA, but not helicase-deficient CsdA mutants. 90 We used csdA deletion cells and screened an E. coli genomic library for protein(s) that can complement the essential CsdA function during cold acclimation. We observed that another DEAD-box RNA helicase, RhlE, complemented the csdA deletion.…”

Helicase Activity Plays a Crucial Role for RNase R Function in Vivo and for RNA Metabolism

“…This scenario would explain the earlier complementation data. What has not been easy to explain is how RNase R lacking its nuclease activity is still able to complement a csdA deletion (11). We have now found in vitro that at high levels, nuclease-deficient RNase R can complement because it converts a duplex substrate to single strands, which then are easily digested by PNPase and RNase R (data not shown).…”

“…We have now found in vitro that at high levels, nuclease-deficient RNase R can complement because it converts a duplex substrate to single strands, which then are easily digested by PNPase and RNase R (data not shown). If a similar situation occurs in vivo, such a mechanism would allow PNPase and the WT RNase R present in the strain to function in the absence of CsdA and lead to the apparent complementation by nuclease-deficient RNase R that was observed (11).…”

“…One aspect of the RNase R helicase activity that is not yet fully understood is its ability to complement the cold shock function of CsdA, a DEAD-box helicase (11). CsdA is essential at low temperatures, and we believe this is because it associates with PNPase and is required for PNPase to function under these conditions (22,23).…”

“…It is known that RNase R can complement the essential function of CsdA, a DEAD-box RNA helicase, during cold shock (11), implying that the helicase activity of RNase R can function in vivo. Moreover, this complementation activity persists even when the nuclease activity of RNase R has been eliminated by mutation.…”