Biochemical and molecular approaches to the study of iron nutrition in the marine cyanobacterium synechococcus WH 7803

Field work with natural phytoplankton assemblages using conventional oceanographic techniques, has established the limited ability of such methods to be able to identify the existence of iron-deplete conditions in the North Atlantic Ocean. Such results confirmed the requirement of "novel" detection methods to determine whether phytoplankton are iron limited in non-productive waters. Carotenoid-containing cell walls from the marine cyanobacterium Synechococcus WH 7803 contained two major polypeptides of Mr - 94,000 and 67,000. The larger of the two appeared to be heat modifiable, a characteristic of porins. Western blot analysis showed that the cell walls from different species of cyanobacteria and one prochlorophyte exhibited a high degree of antigenic relatedness. The effects of iron-deplete conditions upon the outer membrane protein (OMP) profile of Synechococcus WH 7803 was simulated by the addition of 15 nq ml-1 EDDA to normal growth medium. Initial SDS-PAGE analysis revealed the presence of specific iron-regulated outer-membrane proteins. The size of the polypeptides present depended upon the type of iron limitation imposed. Light-stimulated 35S-methionine uptake and incorporation into protein was observed with Synechococcus WH 7803. The reproducibility and sensitivity of this technique resulted in its use as a detection system for the synthesis of iron- regulated membrane proteins. After 21-48 hr growth of Synechococcus WH 7803 cells in iron-deplete medium a protein of Mj. 36,000 was synthesized. Repression of the protein's synthesis occurred after 24 hr growth in iron-replete medium. This protein was localized to both the internal membrane and the cell wall. By contrast, antibodies raised against the 36,000 Da polypeptide were able to detect the protein in internal membrane, cell wall and whole cell fractions of Synechococcus WH 7803 cells grown in both iron-replete and deplete medium. This suggests that the protein is expressed constitutively. Although partial characterization of the 36,000 Da polypeptide has, as yet, been unsuccessful, certain characteristics of the protein were reminiscent of those of TonB. Antibodies raised against a synthetic peptide containing the x-proline dipeptide repeat of TonB were unable to detect a similar protein in Synechococcus WH 7803. However, Southern hybridization analysis using both the S. typhimurium tonb gene and an oligonucleotide probe constructed against the TonB Lys-Pro dipeptide repeat, suggest the presence of a TonB homologue. Once the blockage has been removed from the N-terminus of the 36 kDa protein the sequence should hopefully yield conclusive evidence as to the protein's function. Sequestration of iron by Synechococcus WH 7803 cells was observed using flame atomic absorption spectroscopy on whole cell samples. Such sequestration suggests the presence of iron storage proteins. Western blot analysis and Mossbauer spectroscopy confirmed the presence of an iron storage protein namely bacterioferritin. The 57Fe associated with the storage protein was in the same form as that found in the bacterioferritins of B.coli and P.aeruginosa, as determined by Mossbauer spectroscopy. In addition, siderophore production has not been found in this cyanobacterium.