A novel 14-3-3 gene is osmoregulated in gill epithelium of the euryhaline teleost Fundulus heteroclitus.

We have cloned and analyzed the full-length coding sequence and 3' untranslated region (UTR) of a unique 14-3-3 gene of the euryhaline teleost Fundulus heteroclitus, which we named 14-3-3.a. Phylogenetic analysis of the deduced amino acid sequence revealed that the 14-3-3.a gene product is most similar to vertebrate 14-3-3 zeta and beta, yet it displays considerable divergence to known classes of vertebrate 14-3-3 isoforms. The N and C termini of 14-3-3.a are the most unique regions, whereas the amino acid residues forming the amphipathic ligand-binding groove are highly conserved. F. heteroclitus 14-3-3.a mRNA expression is high in gill epithelium, moderate in intestine and brain, and low in gonads, white muscle and heart. Because 14-3-3 proteins are important molecular scaffolds and cofactors for phosphoproteins and signaling complexes, the high level of 14-3-3.a expression in gill epithelium of the euryhaline teleost F. heteroclitus suggests that it is crucial for signal transduction in gill epithelial cells. We provide evidence that 14-3-3.a is involved in osmosensory signal transduction by showing that its mRNA and protein levels in gill epithelium, but not in any other tissue analyzed, increase two- to fourfold within 24h of salinity transfer of fish from sea water to fresh water. These data are clear evidence for an important role of 14-3-3.a in the remodeling of gill epithelium during transition of euryhaline fish between plasma-hyperosmotic and plasma-hypoosmotic environments.

Mutational analysis of a role for salicylic acid in iron metabolism of Mycobacterium smegmatis.

The role of salicylic acid in iron metabolism was examined in two wild-type strains (mc(2)155 and NCIMB 8548) and three mutant strains (mc(2)1292 [lacking exochelin], SM3 [lacking iron-dependent repressor protein IdeR] and S99 [a salicylate-requiring auxotroph derived in this study]) of Mycobacterium smegmatis. Synthesis of salicylate in SM3 was derepressed even in the presence of iron, as was synthesis of the siderophores exochelin, mycobactin, and carboxymycobactin. S99 was dependent on salicylate for growth and failed to grow with the three ferrisiderophores, suggesting that salicylate fulfills an additional function(s) other than being a precursor of mycobactin and carboxymycobactin. Salicylic acid at 100 microgram/ml repressed the formation of a 29-kDa cell envelope protein (putative exochelin receptor protein) in S99 grown both iron deficiently and iron sufficiently. In contrast, synthesis of this protein was affected only under iron-limited conditions in the parent strain, mc(2)155, and remained unaltered in SM3, suggesting an interaction between the IdeR protein and salicylate. Thus, salicylate may also function as a signal molecule for recognition of cellular iron status. Growth of all strains and mutants with p-aminosalicylate (PAS) at 100 microgram/ml increased salicylate accumulation between three- and eightfold under both iron-limited and iron-sufficient growth conditions and decreased mycobactin accumulation by 40 to 80% but increased carboxymycobactin accumulation by 50 to 55%. Thus, although PAS inhibited salicylate conversion to mycobactin, presumptively by blocking salicylate AMP kinase, PAS also interferes with the additional functions of salicylate, as its effect was heightened in S99 when the salicylate concentration was minimal.