No abstract

SummaryLike other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15ЊC as well as at 37ЊC, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37ЊC and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B. subtilis. After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.

Bacteria respond to a decrease in temperature with the induction of proteins that are classified as coldinduced proteins (CIPs). Using two-dimensional gel electrophoresis, we analyzed the cold shock response in Bacillus subtilis. After a shift from 37 to 15؇C, the synthesis of a majority of proteins was repressed; in contrast, 37 proteins were synthesized at rates higher than preshift rates. One hour after cold shock, the induction of CIPs decreased, and after 2 h, general protein synthesis resumed. The identified main CIPs were excised from two-dimensional gels and were subjected to microsequencing. Three small acidic proteins that showed the highest relative induction after cold shock were highly homologous and belonged to a protein family of which one member, the major cold shock protein, CspB, has previously been characterized. Two-dimensional gel analyses of a cspB null mutant revealed that CspB affects the level of induction of several CIPs. Other identified CIPs function at various levels of cellular physiology, such as chemotaxis (CheY), sugar uptake (Hpr), translation (ribosomal proteins S6 and L7/L12), protein folding (PPiB), and general metabolism (CysK, IlvC, Gap, and triosephosphate isomerase).Bacteria must adapt to continuous changes in the environment, such as changes in the availability of nutrients or oxygen. Invariably, the response to drastic chemical and physical changes in the surroundings involves the induction of sets of specific proteins. This has been shown for various environmental stresses, including an increased salt concentration, a rise in temperature (heat shock), phage infection, or ethanol treatment (1a, 12, 17, 56). The functions and regulation of some stress-induced proteins, particularly the well-characterized heat shock proteins that act as molecular chaperones (61), have been elucidated. However, little is known about the functions of proteins induced after a decrease in temperature. A cold shock response has been found in Escherichia coli (25), and the induction of proteins in response to cold shock was monitored in Bacillus (30,58,60), Listeria (41), and Rhizobium (10) species. In E. coli, the number of proteins synthesized decreases drastically after a shift from 37 to 10ЊC, and cellular growth arrests. After 2 h, only 28 proteins are detectably produced, 14 at rates higher than preshift rates. Three hours after a cold shock, the number of proteins increases again, until after 4 h, normal protein synthesis and growth are resumed. Cold-induced proteins (CIPs) include mainly transcriptional and translational proteins, components of the pyruvate dehydrogenase complex, and CspA, the only protein that is not present at 37ЊC. Most CIPs are synthesized at a 2-to 10-fold higher level compared with preshift levels, in contrast to CspA, which is induced about 200-fold (25). Other proteins that are continually synthesized after a cold shock have been identified as ribosomal proteins L7, L12, S1, S6B, and S6A, trigger factor, elongation factor Tu (EF-Tu), EF-Ts, EF-G, and the ␤ subun...

The catalysis of bioorthogonal transformations inside living organisms is a formidable challenge--yet bears great potential for future applications in chemical biology and medicinal chemistry. We herein disclose highly active organometallic ruthenium complexes for bioorthogonal catalysis under biologically relevant conditions and inside living cells. The catalysts uncage allyl carbamate protected amines with unprecedented high turnover numbers of up to 270 cycles in the presence of water, air, and millimolar concentrations of thiols. By live-cell imaging of HeLa cells and with the aid of a caged fluorescent probe we could reveal a rapid development of intense fluorescence within the cellular cytoplasm and therefore support the proposed bioorthogonality of the catalysts. In addition, to illustrate the manifold applications of bioorthogonal catalysis, we developed a method for catalytic in-cell activation of a caged anticancer drug, which efficiently induced apoptosis in HeLa cells.

The cold-shock domain (CSD) is found in many eukaryotic transcriptional factors and is responsible for the specific binding to DNA of a cis-element called the Y-box. The same domain exists in the sequence of the Xenopus RNA-binding proteins FRG Y1 and FRG Y2 (refs 1, 3). The major cold-shock proteins of Escherichia coli (CS7.4) and B. subtilis (CspB) have sequences that are more than 40 per cent identical to the cold-shock domain. We present here the three-dimensional structure of CspB determined by nuclear magnetic resonance spectroscopy. The 67-residue protein consists of an antiparallel five-stranded beta-barrel with strands connected by turns and loops. The structure resembles that of staphylococcal nuclease and the gene-5 single-stranded-DNA-binding protein. A three-stranded beta-sheet, which contains the conserved RNA-binding motif RNP1 as well as a motif similar to RNP2 in two neighbouring antiparallel beta-strands, has basic and aromatic residues at its surface which could serve as a binding site for single-stranded DNA. CspB binds to single-stranded DNA in gel retardation experiments.