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.

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The presence of the (Gly-Xaa-Yaa) n open reading frames in different bacteria predicts the existence of an expanded family of collagen-like proteins. To further explore the triple-helix motif and stabilization mechanisms in the absence of hydroxyproline (Hyp), predicted novel collagen-like proteins from Gram-positive and -negative bacteria were expressed in Escherichia coli and characterized. Soluble proteins capable of successful folding and in vitro refolding were observed for collagen proteins from Methylobacterium sp 4-46, Rhodopseudomonas palustris and Solibacter usitatus. In contrast, all protein constructs from Clostridium perfringens were found predominantly in inclusion bodies. However, attachment of a heterologous N-terminal or C-terminal noncollagenous folding domain induced the Clostridium perfringens collagen domain to fold and become soluble. The soluble constructs from different bacteria had typical collagen triple-helical features and showed surprisingly similar thermal stabilities despite diverse amino acid compositions. These collagen-like proteins provide a resource for the development of biomaterials with new properties.

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