The lack of a stress response in Hydra oligactis is due to reduced hsp70 mRNA stability.

Synthesis and degradation of hsp70 mRNA was examined and compared in Hydra species living in different habitats and showing different heat-shock response. Hydra oligactis is restricted to habitats of low temperature and relatively stable pH. We have shown previously that this species is unable to acquire thermotolerance [Bosch, T., Krylow, S., Bode, H. & Steele, R. (1988) Proc. Natl. Acad. Sci. USA 85, 7927-7931] and synthesizes significantly less heat-shock protein and hsp70 mRNA [Gellner, K., Praetzel, G. & Bosch, T. C. G. (1992) Eur J. Biochem. 210, 683-691] in response to stress than related species, such as Hydra bulgaris or Hydra magnipapillata, which are adapted to habitats of wide temperature range and variable water quality. To examine the mechanisms responsible for the differential heat-shock responses in these species, a construct containing H. magnipapillata hsp70 regulatory sequences fused to firefly luciferase was introduced into H. oligactis and H. magnipapillata polyps, and expression of luciferase examined. The results showed that luciferase can be expressed equally well in a heat-inducible manner in both species, suggesting that H. oligactis heat-shock factor can interact with H. magnipapillata heat-shock elements. Northern blots of alpha-amanitin-treated polyps demonstrated that the half-life of hsp70 mRNA in heat-shocked H. oligactis is drastically shorter than in H. magnipapillata. Thus, differences in hsp70 mRNA stability appear to be responsible for the habitat-correlated differences in the stress response in Hydra species.

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE.

Homopolymeric alpha-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5' end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.