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The rpoS (katF) gene of Escherichia coli encodes a putative sigma factor (ajS) required for the expression of a variety of stationary phase-induced genes, for the development of stationary-phase stress resistance, and for long-term starvation survival (R. Lange and R. Hengge-Aronis, Mol. Microbiol. 5:49-59, 1991). Here we show that the genes otsA, otsB, treA, and osmB, previously known to be osmotically regulated, are also induced during transition into stationary phase in a rs-dependent manner. otsA and otsB, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively, are involved in os-dependent stationary-phase thermotolerance. Neither os nor trehalose, however, is required for the development of adaptive thermotolerance in growing cells, which might be controlled by cJE.

Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITreHIlI c)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EfiTre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate-and HTre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.The synthesis of internal trehalose in Escherichia coli in response to high osmolarity has been studied in detail on a genetic and biochemical level (12, 30), yet little is known about trehalose transport and metabolism. Early reports have described E. coli mutants that were partially defective in the utilization of trehalose. The mutations mapped at 26 min on the linkage map (3; for Salmonella typhimurium, see reference 29). Marechal (20) later reported the existence of a specific enzyme II of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) by demonstrating the PEP-dependent phosphorylation of trehalose. He also claimed, on the basis of biochemical studies, the existence of an enzyme able to hydrolyze trehalose-6-phosphate to glucose-6-phosphate and glucose (20). Different results were obtained by Postma et al. (23), who reported that trehalose is transported in S. typhimurium via the mannose-PTS without phosphorylation. Both studies reported the existence of a trehalose-inducible trehalase in crude extracts.A periplasmic trehalase was subsequently discovered and purified from E. coli, and mutants, termed treA, were isolated that lacked this enzyme. treA was mapped at 26 min (5), and the treA gene was cloned, sequenced, and found to be the only gene in the operon (14). Periplasmic trehalase synthesis is not induced by trehalose but rather by growth in the presence of 250 mM NaCl (5). Apparently, the function of the periplasmic trehalase is to ensure the utilization of trehalose under conditions of high osmolarity...

Bacillus subtilis has developed sophisticated mechanisms to withstand fluctuations in temperature. Membrane fatty acids are the major determinants for a sufficiently fluid membrane state to ensure the membrane’s function at all temperatures. The fatty acid profile of B. subtilis is characterized by a high content of branched fatty acids irrespective of the growth medium. Here, we report on the importance of isoleucine for B. subtilis to survive cold shock from 37 to 15°C. Cold shock experiments with strain JH642 revealed a cold-protective function for all intermediates of anteiso-branched fatty acid biosynthesis. Metabolites related to iso-branched or straight-chain fatty acid biosynthesis were not protective. Fatty acid profiles of differentB. subtilis wild-type strains proved the altered branching pattern by an increase in the anteiso-branched fatty acid content and a concomitant decrease of iso-branched species during cold shock. There were no significant changes in the fatty acid saturation or acyl chain length. The cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine correlated with their inability to synthesize more anteiso-branched fatty acids, as shown by the fatty acid profile. The switch to a fatty acid profile dominated by anteiso-C15:0 and C17:0 at low temperatures and the cold-sensitive phenotype of isoleucine-deficient strains in the absence of isoleucine focused our attention on the critical role of anteiso-branched fatty acids in the growth of B. subtilisin the cold.

p21 CIP1/WAF1 is an inhibitor of cyclin-dependent kinases and, in normal tissues including squamous epithelia, has been associated with cell-cycle exit and differentiation. As shown in this pilot study, however, the majority of head-and-neck squamous-cell carcinomas (HNSCC) display aberrant p21 CIP1/WAF1 expression: of 42 tumors analyzed by immunohistochemical staining, 28 (67%) over-expressed the p21 CIP1/WAF1 protein. Accumulation of p21 CIP1/WAF1 was independent of the histological grade of the tumors as well as the genetic status of the p53 gene. In many cases, most notably in poorly differentiated or undifferentiated HNSCC, p21 CIP1/WAF1 -positive cells were actively proliferating tumor cells, since they also expressed proliferating-cell nuclear antigen (PCNA) and Ki-67. Accumulation of p21 CIP1/WAF1 occurred through a posttranscriptional mechanism since, in contrast to immunohistochemical analysis of the p21 CIP1/WAF1 protein, in situ hybridization showed no increase of mRNA levels as compared with cells in normal mucosa (n 5 25). Clinically, among the patients with p21 CIP1/WAF1 -over-expressing tumors, there was increased recurring disease (p 5 0.03; x 2 -test), shortened disease-free survival (p 5 0.0019; log-rank test) and shortened overall survival (p 5 0.0071; log-rank test). These in vivo data indicate that in many HNSCC, accumulated p21 CIP1/WAF1 is compatible with increased tumor-cell proliferation, and they provide preliminary evidence that p21 CIP1/WAF1 may be of prognostic and predictive significance. Int. J. Cancer 74:383-389, 1997.r 1997 Wiley-Liss, Inc. p21 CIP1/WAF1 has been shown to inhibit the activity of several cyclin/cyclin-dependent kinase complexes and to block cell-cycle progression (Harper et al., 1993;Xiong et al., 1993;Gu et al., 1993). It was identified as a gene whose product was transcriptionally activated by wild-type but not mutant p53 protein, serving as a mediator of the cell-cycle-arrest function of p53 (El-Deiry et al., 1993). Simultaneously, it was found to be associated with senescence (Noda et al., 1994) and terminal differentiation (Jiang et al., 1994;Steinman et al., 1994; Halevy et al., 1995). These properties made the p21 CIP1/WAF1 gene a prominent candidate tumorsuppressor gene. However, extensive searches failed to reveal genetic alterations of the gene in human tumors, except for the rare occurrence of polymorphisms (Shiohara et al., 1994).The precise role of p21 CIP1/WAF1 in the control of proliferation and differentiation is not clear. During mouse embryonic development, p21 CIP1/WAF1 mRNA expression was found in the post-mitotic cells of many tissues, and this expression occurred independently of p53. This suggested that p21 CIP1/WAF1 provides a tight link between cell-cycle arrest and exit and differentiation (Parker et al., 1995). However, knockout mice lacking a functional p21 CIP1/WAF1 developed fairly normally, indicating that the presumed function of p21 CIP1/WAF1 in differentiation was redundant (Deng et al., 1995). In addition, in contrast to p53 kno...