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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Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.

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Proper folding of the (Gly-Xaa-Yaa) n sequence of animal collagens requires adjacent N-or C-terminal noncollagenous trimerization domains which often contain coiled-coil or beta sheet structure. Collagen-like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen-like protein from Streptococcus pyogenes has an N-terminal globular domain, designated V sp , adjacent to its triple-helix domain. The V sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V sp domain alone is shown to form trimers with a significant a-helix content and to have a thermal stability of T m 5 45°C. Examination of a new construct shows that the V sp domain facilitates efficient in vitro refolding only when it is located N-terminal to the triple-helix domain but not when C-terminal to the triple-helix domain. Fusion of the V sp domain N-terminal to a heterologous (GlyXaa-Yaa) n sequence from Clostridium perfringens led to correct folding and refolding of this triplehelix, which was unable to fold into a triple-helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly-XaaYaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple-helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.